Inhibitors of Late SV40 Factor (LSF) as cancer chemotherapeutics

ABSTRACT

The present invention is directed to methods, compositions and kits for treatment of cancer, e.g. heptacellular carcinoma. In some embodiments, the present invention discloses the use of a small-molecule compound of formula (I)-(XXVI) and (III′) as disclosed herein to inhibit transcription factor Late SV40 Factor (LSF) for treatment of cancer, e.g., HCC.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 15/389,564 filed Dec. 23, 2016, which is acontinuation-in-part of and claims priority to U.S. patent applicationSer. No. 15/337,584, filed Oct. 28, 2016, which is acontinuation-in-part of and claims priority to U.S. patent applicationSer. No. 13/879,106, filed Aug. 20, 2013, now U.S. Pat. No. 9,597,325issued Mar. 21, 2017, which is a 35 U.S.C. § 371 National Phase EntryApplication of International Application No. PCT/US2011/054305, filedSep. 30, 2011, which designates the U.S., and which benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application Ser. No. 61/392,607filed on Oct. 13, 2010, the contents of all which are incorporatedherein by reference in their entirety. This application is acontinuation of and claims priority to U.S. patent application Ser. No.15/337,584, filed Oct. 28, 2016, which is a continuation-in-part of andclaims priority to U.S. patent application Ser. No. 13/879,106, filedAug. 20, 2013, now U.S. Pat. No. 9,597,325 issued Mar. 21, 2017, whichis a 35 U.S.C. § 371 National Phase Entry Application of InternationalApplication No. PCT/US2011/054305, filed Sep. 30, 2011, which designatesthe U.S., and which benefit under 35 U.S.C. 119(e) of U.S. ProvisionalPatent Application Ser. No. 61/392,607 filed on Oct. 13, 2010, thecontents of all which are incorporated herein by reference in theirentirety.

GOVERNMENT SUPPORT

This invention was made in part with U.S. Government support from theNational Institutes of Health grant P50 GM067041 and the NationalScience Foundation HRD-0820175. The U.S. Government has certain rightsin this application.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 25, 2017, isnamed 701586-068372_SequenceListing.txt and is 9,976 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to methods, compositions andkits for treatment of cancer, e.g. hepatocellular carcinoma (HCC). Insome embodiments, the invention relates to the use of small-moleculecompounds to inhibit expression of the transcription factor Late SV40Factor (LSF) for treatment of cancer, e.g., HCC.

BACKGROUND OF THE INVENTION

Hepatocellular carcinoma (HCC) is a primary malignant tumor, whichdevelops in the liver. HCC is one of the five most common cancers andthe third leading cause of cancer deaths worldwide. The incidence of HCCis increasing despite a decrease in overall incidence of all cancers. Inthe United States, the estimated new cases of HCC for 2008 were 21,370,of which 18,410 were expected to die. There are multiple etiologies,with subcategories displaying distinct gene expression profiles. Theprognosis of HCC remains poor. The mean 5-year survival rate is lessthan 10%. The mortality rate of HCC parallels that of its incidencebecause HCC is a tumor with rapid growth and early vascular invasionthat is resistant to conventional chemotherapy, and only a singlesystemic therapy (sorafenib) is available for advanced disease, althoughthe survival benefit averages only a few months.

Hepatocellular carcinoma (HCC) is characterized by late stage diagnosisand a poor prognosis for treatment, usually consisting of surgicalresection of the tumor and chemotherapy¹⁻³. Currently, the only approvedtreatment for primary malignancies is sorafenib, a receptor tyrosinekinase and Raf inhibitor originally developed for primary kidney cancerthat is also marginally effective against HCC, increasing survival by2-3 months as a single treatment.

The current treatment options for HCC are not optimal, especiallyfollowing metastasis. Irradiation and chemotherapies have not so farproved to be satisfactory; surgery is the most effective treatment ofHCC. However, surgery is only appropriate for patients with smallresectable tumors. Only a single, molecularly based drug (Sorafenib),which targets tyrosine kinase receptors and the MEK/ERK pathway, hasgenerated responses in patients as a single therapy. However, increasedsurvival times with this drug are only a few months. As such, it isimperative to discover novel, effective, and targeted therapies for thishighly aggressive cancer. In particular, there is a strong need in theart for improved methods for treatment of HCC with small-molecule drugs.

The transcription factor LSF, a member of a small family oftranscription factors conserved throughout the animal kingdom⁹, isubiquitously expressed in mammalian tissues and cell lines¹⁰. LSFactivity is tightly controlled as cells progress from quiescence intoDNA replication (G0 to 5)^(11,12), and it is required for efficientprogression of cells through the G1/S transition^(4,5). Regulation ofLSF activity normally occurs via post-translational modifications, withLSF protein levels generally being low and constant. However, LSFprotein levels were recently shown to be highly upregulated in tumorcells, particularly in HCC cell lines and HCC patient samples^(6,13).These elevated LSF levels were shown to promote oncogenesis in the HCCcells.

SUMMARY OF THE INVENTION

The present invention is generally directed to methods, compositions andkits to treat cancer, e.g. hepatocellular carcinoma (HCC), for example,by using inhibitors of late SV40 factor (LSF), such as a compoundrepresented by formula (I) as disclosed herein. In some embodiments, aninhibitor of LSF is a compound of formula (IV). In some embodiments, aninhibitor of LSF is any one of compounds of formula (IV) to (XXVI). Insome embodiments, the LSF inhibitor compounds, of formulas (I) to (XXVI)as disclosed herein can be used to treat other cancers, for example,cervical cancer, colon cancers, melanomas and the like.

Recently, the inventors were involved in demonstrating that thetranscription factor LSF, which was previously reported to mediate G1/Sprogression in multiple cell types^(4,5), can function as an oncogenefor HCC⁶. Small molecule inhibitors of oncogenic kinases can providetremendous therapeutic benefit, due to the phenomenon of oncogeneaddiction^(7,8), in which primary cancers are uniquely dependent on anoncogene for continued cell growth and survival.

Previously, the inventors in collaboration with other scientists havereported that the expression of LSF is up-regulated in subjects, e.g.human subjects, with HCC (see Yoo et al., PNAS, 2010; 107; 8357-8362,which is incorporated herein in its entirety by reference). Herein, theinventors have discovered that small-molecule compounds disclosed hereinthat are specific inhibitors of LSF, and can cause cell death in HCCcell lines, as well as in primary cancer cells and cell lines derivedfrom other cancer types. Previously, the inventors have demonstrated incollaboration with other scientists that the small molecules asdisclosed herein decreases tumorigenesis and metastasis of HCC in an invivo mouse model of HCC. Accordingly, the inventors have discovered afamily of small molecule inhibitors LSF as disclosed herein, and can beused as chemotherapeutics agents for treatment of cancers, e.g., HCC insubjects, such as human subjects.

Accordingly, the inventors demonstrate herein that small molecules thatspecifically inhibit LSF could provide treatment for cancers, e.g., HCC.In particular, the inventors have identified a family of small moleculesthat specifically target the DNA-binding and correspondingtranscriptional activities of LSF, and that inhibit proliferation of anumber of cancer cell lines, e.g., cervical cancer cell lines, coloncancer cell lines, breast cancer cell lines, endometrium cell lines,kidney cell lines, melanoma cell lines, etc. The LSF inhibitors asdisclosed herein rapidly induce apoptosis in an aggressive HCC cell linein vitro. Additionally, in collaboration with other scientists, theinventors have demonstrated that the small molecule inhibitors of LSF asdisclosed herein significantly inhibits tumor growth in a mousexenograft model, with no observable tissue toxicity (data not shown). Incontrast, the inventors have demonstrated that the viability of primarycells is unaffected in vitro (data not shown). These data demonstrateoncogene addiction of HCC cells to LSF and support the feasibility ofdirectly targeting this transcription factor for chemotherapeuticintervention.

One aspect of the present invention relates to a method to inhibit LSFin a subject, for example, using a compound of formula (I), orenantiomers, prodrugs, derivatives, or pharmaceutically acceptable saltsthereof, wherein the formula (I) has the structure:

wherein:

R¹, R², R³ and R⁴ are each independently selected from the group ofhydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl, OR^(3A),SR^(3A), SO₂R^(3A), NR^(3A)R^(4A), PO_(m)(R^(3A))_(n), F, Br, Cl, I,heteroaryl, or aryl, wherein the alkyl, haloalkyl, heteroalkyl,heteroaryl, and aryl can be optionally substituted with halogen, C₁-C₄alkyl, C₁-C₄ haloalkyl, C₁-C₄ heteroalkyl, di(C₁-C₂₄alkyl)amino, orC₁-C₆ alkoxy.

In some embodiments, R² and R³ together form a second bond between thecarbons to which they are attached.

R⁵ is selected from the group of hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl,C₁-C₆ heteroalkyl, aryl, heteroaryl, F, Br, Cl, or I.

R⁶ and R⁷ are each independently selected from the group of hydrogen, F,Br, Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A), SO₂R^(3A),PO_(m)(R^(3A))_(n), C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,aryl or heteroaryl.

R⁸ and R⁹ are each independently selected from the group of hydrogen,C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ heteroalkyl, OR^(3A), SR^(3A),SO₂R^(3A), NR^(3A)R^(4A), PO_(m)(R^(3A))_(n), F, Br, Cl, I, heteroarylor aryl, provided R⁹ is not NR^(3A)R^(4A).

In some embodiments, R⁸ and R⁹ together with the carbons to which theyare attached form an optionally substituted 5-8 membered cycloalkyl orheterocycyl.

R^(3A) and R^(4A) are each independently selected from the group ofhydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl,C₁-C₈ heteroalkyl, heteroaryl, or aryl, wherein the alkyl, alkenyl,alkynyl, haloalkyl, heteroalkyl, heteroaryl, and aryl can be optionallysubstituted with halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄heteroalkyl or C₁-C₆ alkoxy.

m is 1, 2, or 3; and n is 1, 2, or 3;

In one embodiment, the subject suffers from or is at risk ofhepatocellular carcinoma.

Another aspect of the invention relates to methods and compositions totreat hepatocellular carcinoma in a subject. The methods of theinvention as disclosed herein include administration to the subject apharmaceutical composition comprising a compound of formula (I) asdisclosed herein, or enantiomers, prodrugs, derivatives orpharmaceutically acceptable salts thereof.

In some embodiments, a compound of the invention as disclosed herein canbe used in combination therapy.

In various embodiments, a compound of formula (I) can be represented byformula (II), wherein the formula (II) has the structure:

In formula (II), R¹, R², R³ and R⁴ are each independently selected fromthe group of hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,OR^(3A), SR^(3A), SO₂R^(3A), NR^(3A)R^(4A), PO_(m)(R^(3A))_(n), F, Br,Cl, I, heteroaryl, or aryl, wherein the alkyl, haloalkyl, heteroalkyl,heteroaryl, and aryl can be optionally substituted with halogen, C₁-C₄alkyl, C₁-C₄ haloalkyl, C₁-C₄ heteroalkyl, di(C₁-C₂₄alkyl)amino, orC₁-C₆ alkoxy.

In some embodiments, R^(3A) is C₁-C₈ alkyl. In some embodiments, C₁-C₈alkyl is a straight chain alkyl. In some embodiments, C₁-C₈ alkyl is abranched chain alkyl. In some embodiments, C₁-C₈ alkyl is methyl, ethyl,n-propyl, i-propyl, n-butyl, i-butyl, or t-butyl.

In some embodiments, OR^(3A) is O—C₁-C₈ alkyl. In some embodiments,C₁-C₈ alkyl is a straight chain alkyl. In some embodiments, C₁-C₈ alkylis a branched chain alkyl. In some embodiments, C₁-C₈ alkyl is methyl,ethyl, n-propyl, i-propyl, n-butyl, i-butyl, or t-butyl.

R⁵ is selected from the group of hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl,C₁-C₆ heteroalkyl, aryl, heteroaryl, F, Br, Cl, or I.

R⁶ and R⁷ are each independently selected from the group of hydrogen, F,Br, Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A), SO₂R^(3A),PO_(m)(R^(3A))_(n), C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,aryl or heteroaryl. In some embodiments, R⁷ is OR^(3A). In someembodiments, OR^(3A) is O—C₁-C₈ alkyl. In some embodiments, C₁-C₈ alkylis a branched chain alkyl. In some embodiments, C₁-C₈ alkyl is methyl,ethyl, n-propyl, propyl, n-butyl, i-butyl, or t-butyl.

R⁸ is selected from the group of hydrogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl,C₁-C₄ heteroalkyl, OR^(3A), SR^(3A), SO₂R^(3A), NR^(3A)R^(4A),PO_(m)(R^(3A))_(n), F, Br, Cl, I, heteroaryl or aryl. In someembodiments, R⁸ is OR^(3A). In some embodiments, OR^(3A) is O—C₁-C₈alkyl. In some embodiments, C₁-C₈ alkyl is a branched chain alkyl. Insome embodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, i-propyl,n-butyl, i-butyl, or t-butyl.

R^(3A) and R^(4A) are each independently selected from the group ofhydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl,C₁-C₈ heteroalkyl, heteroaryl, or aryl, wherein the alkyl, alkenyl,alkynyl, haloalkyl, heteroalkyl, heteroaryl, and aryl can be optionallysubstituted with halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄heteroalkyl or C₁-C₆ alkoxy.

In some embodiments, R² and R³ together form a second bond between thecarbons to which they are attached. In some embodiments, R² is asubstituted aryl. In some embodiments, R² is a substituted phenyl. Insome embodiments, the substituted phenyl is a C₁-C₆ alkoxy substitutedphenyl. In certain embodiments, C₁-C₆ alkoxy is OMe, OEt, O^(n)Pr,O^(i)Pr, O^(n)Bu, O^(i)Bu, or O^(t)Bu.

In some embodiments, R⁸ and OR^(3A) together with the carbons to whichthey are attached form an optionally substituted 5-8 membered cycloalkylor heterocycyl.

m is 1, 2, or 3; and n is 1, 2, or 3;

In some embodiments, inhibitors of LSF with formula (I) can berepresented by formula (III), wherein the formula (III) has thestructure:

In formula (III), R¹, R², R³ and R⁴ are each independently selected fromthe group of hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,OR^(3A), SR^(3A), SO₂R^(3A), NR^(3A)R^(4A), PO_(m)(R^(3A))_(n), F, Br,Cl, I, heteroaryl, or aryl, wherein the alkyl, haloalkyl, heteroalkyl,heteroaryl, and aryl can be optionally substituted with halogen, C₁-C₄alkyl, C₁-C₄ haloalkyl, C₁-C₄ heteroalkyl, di(C₁-C₂₄alkyl)amino, orC₁-C₆ alkoxy.

In some embodiments, R² and R³ together form a second bond between thecarbons to which they are attached.

R⁵ is selected from the group of hydrogen, C₁-C₆ alkyl, C₁-C₆haloalkyl,C₁-C₆ heteroalkyl, aryl, heteroaryl, F, Br, Cl, or I;

R⁶ and R⁷ are each independently selected from the group of hydrogen, F,Br, Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A), SO₂R^(3A),PO_(m)(R^(3A))_(n), C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,aryl or heteroaryl;

R¹⁰ and R¹¹ are each independently selected from the group of hydrogen,F, Br, Cl, or I.

R^(3A) and R^(4A) are each independently selected from the group ofhydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl,C₁-C₈ heteroalkyl, heteroaryl, or aryl, wherein the alkyl, alkenyl,alkynyl, haloalkyl, heteroalkyl, heteroaryl, and aryl can be optionallysubstituted with halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄heteroalkyl or C₁-C₆ alkoxy.

m is 1, 2, or 3; and n is 1, 2, or 3;

In some embodiments, inhibitors of LSF with formula (III) can berepresented by formula (XXVI), wherein the formula (XXVI) has thestructure:

In formula (XXVI), R², R³ and R⁴ are each independently selected fromthe group of hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,OR^(3A), SR^(3A), SO₂R^(3A), NR^(3A)R^(4A), PO_(m)(R^(3A))_(n), F, Br,Cl, I, heteroaryl, or aryl, wherein the alkyl, haloalkyl, heteroalkyl,heteroaryl, and aryl can be optionally substituted with halogen, C₁-C₄alkyl, C₁-C₄ haloalkyl, C₁-C₄ heteroalkyl, di(C₁-C₂₄alkyl)amino, orC₁-C₆ alkoxy. In various embodiments, R² and R³ together can form asecond bond between the carbons to which they are attached.

R⁵ is selected from the group of hydrogen, C₁-C₆ alkyl, C₁-C₆haloalkyl,C₁-C₆ heteroalkyl, aryl, heteroaryl, F, Br, Cl, or I;

R⁶, R⁷, R¹³, R¹⁴, R¹⁵ and R^(15′) are each independently selected fromthe group of hydrogen, F, Br, Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A),SO₂R^(3A), PO_(m)(R^(3A))_(n), C₁-C₆ alkyl, C₂-C₈ alkenyl, C₁-C₆haloalkyl, C₁-C₆ heteroalkyl, aryl or heteroaryl;

each R¹² is independently selected from the group of hydrogen, F, Br,Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A), SO₂R^(3A), PO_(m)(R^(3A))_(n),C₁-C₆ alkyl, C₂-C₈ alkenyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl, aryl orheteroaryl;

R¹⁶ and R¹⁷ are each independently selected from the group of hydrogen,F, Br, Cl, I, C₁-C₄ alkyl, or OR^(3A);

R^(3A) and R^(4A) are each independently selected from the group ofhydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl,C₁-C₈ heteroalkyl, heteroaryl, or aryl, wherein the alkyl, alkenyl,alkynyl, haloalkyl, heteroalkyl, heteroaryl, and aryl can be optionallysubstituted with halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄heteroalkyl or C₁-C₆ alkoxy.

m is 1, 2, or 3; and n is 1, 2, or 3.

In some embodiments, at least two of R¹², R¹³, R¹⁴, R¹⁵ and R^(15′) arenot hydrogen. In some embodiments, R¹² and at least one of R¹⁵ andR^(15′) are not hydrogen.

In some embodiments, at least one of R¹², R¹³, R¹⁴, R¹⁵ and R^(15′) isOR^(3A) and at least one of R¹², R¹³, R¹⁴, R¹⁵ and R^(15′) is F, Br, Cl,I, OR^(3A), NR^(3A)R^(4A), SR^(3A), SO₂R^(3A), PO_(m)(R^(3A))_(n), C₁-C₆alkyl, C₂-C₈ alkenyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl, aryl orheteroaryl.

In some embodiments, at least one of R¹², R¹³, R¹⁴, R¹⁵ and R^(15′) isOR^(3A) and at least one of R¹², R¹³, R¹⁴, R¹⁵ and R^(15′) is F, Br, Cl,I, NR^(3A)R^(4A), SO₂R^(3A), PO_(m)(R^(3A))_(n), C₂-C₈ alkenyl, C₁-C₆haloalkyl or C₁-C₆ heteroalkyl.

In some embodiments, at least one of R¹², R¹³, R¹⁴, R¹⁵ and R^(15′) isOR^(3A) and at least one of R¹², R¹³, R¹⁴, R¹⁵ and R^(15′) is F, Br, Cl,NR^(3A)R^(4A) or C₂-C₈ alkenyl.

In some embodiments, at least one of R¹², R¹⁵ and R^(15′) is OR^(3A) andat least one of R¹², R¹⁵ and R^(15′) is F, Br, Cl, I, OR^(3A),NR^(3A)R^(4A), SR^(3A), SO₂R^(3A), PO_(m)(R^(3A))_(n), C₁-C₆ alkyl,C₂-C₈ alkenyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl, aryl or heteroaryl.

In some embodiments, at least one of R¹², R¹⁵ and R^(15′) is OR^(3A) andat least one of R¹², R¹⁵ and R^(15′) is F, Br, Cl, I, NR^(3A)R^(4A),SO₂R^(3A), PO_(m)(R^(3A))_(n), C₂-C₈ alkenyl, C₁-C₆ haloalkyl or C₁-C₆heteroalkyl.

In some embodiments, at least one of R¹², R¹⁵ and R^(15′) is OR^(3A) andat least one of R¹², R¹⁵ and R^(15′) is F, Br, Cl, NR^(3A)R^(4A) orC₂-C₈ alkenyl.

In some embodiments, at least one of R¹⁵ and R^(15′) is OR^(3A) and R¹²is F, Br, Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A), SO₂R^(3A),PO_(m)(R^(3A))_(n), C₁-C₆ alkyl, C₂-C₈ alkenyl, C₁-C₆ haloalkyl, C₁-C₆heteroalkyl, aryl or heteroaryl.

In some embodiments, at least one of R¹⁵ and R^(15′) is OR^(3A) and R¹²is F, Br, Cl, I, NR^(3A)R^(4A), SO₂R^(3A), PO_(m)(R^(3A))_(n), C₂-C₈alkenyl, C₁-C₆ haloalkyl or C₁-C₆ heteroalkyl.

In some embodiments, at least one of R¹⁵ and R^(15′) is OR^(3A) and R¹²is F, Br, Cl, NR^(3A)R^(4A) or C₂-C₈ alkenyl.

In some embodiments, R¹⁵ or R^(15′) is OR^(3A) and R¹² is F, Br, Cl, I,OR^(3A), NR^(3A)R^(4A), SR^(3A), SO₂R^(3A), PO_(m)(R^(3A))_(n), C₁-C₆alkyl, C₂-C₈ alkenyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl, aryl orheteroaryl.

In some embodiments, R¹⁵ or R^(15′) is OR^(3A) and R¹² is F, Br, Cl, I,NR^(3A)R^(4A), SO₂R^(3A), PO_(m)(R^(3A))_(n), C₂-C₈ alkenyl, C₁-C₆haloalkyl or C₁-C₆ heteroalkyl.

In some embodiments, R¹⁵ or R^(15′) is OR^(3A) and R¹² is F, Br, Cl,NR^(3A)R^(4A) or C₂-C₈ alkenyl.

In some embodiments, one of R¹⁵ and R^(15′) is hydrogen and the other isOR^(3A).

In some embodiments, one of R¹⁵ and R^(15′) is hydrogen and the other isOR^(3A), and R¹² is F, Br, Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A),SO₂R^(3A), PO_(m)(R^(3A))_(n), C₁-C₆ alkyl, C₂-C₈ alkenyl, C₁-C₆haloalkyl, C₁-C₆ heteroalkyl, aryl or heteroaryl.

In some embodiments, one of R¹⁵ and R^(15′) is hydrogen and the other isOR^(3A), and R¹² is F, Br, Cl, I, NR^(3A)R^(4A), SO₂R^(3A),PO_(m)(R^(3A))_(n), C₂-C₈ alkenyl, C₁-C₆ haloalkyl or C₁-C₆ heteroalkyl.

In some embodiments, one of R¹⁵ and R^(15′) is hydrogen and the other isOR^(3A), and R¹² is F, Br, Cl, NR^(3A)R^(4A) or C₂-C₈ alkenyl.

In some embodiments, R¹³, R¹⁴ and one of R¹⁵ and R^(15′) is hydrogen.

In some embodiments, R², R³ and R⁴ are hydrogen or R⁴ is hydrogen and R²and R³ together form a second bond between the carbons to which they areattached.

In some embodiments, R⁵ is hydrogen or C₁-C₆alkyl. Exemplary alkyls forR⁵ include, but are not limited to methyl, ethyl, propyl, isopropyl,1-butyl, 2-butyl, 2-methylpropyl, pentyl, t-butyl, and hexyl. In someembodiments, R⁵ is ethyl.

In some embodiments, R⁶ and R⁷ both are hydrogen.

In some embodiments, R¹⁶ and R¹⁷ are independently hydrogen, F, Br, Clor I. In some embodiments R¹⁶ and R¹⁷ both are hydrogen, F, Br or Cl.

In some embodiments, the inhibitor of LSF is a compound of Formula(III′):

or enantiomers, prodrugs, derivatives, and pharmaceutically acceptablesalts thereof.

In compounds of Formula (III′), R^(1′) is an aryl substituted with atleast one C₁-C₆ alkoxyl and at least one di(C₁-C₂₄alkyl)amino, halogen,C₁-C₆alkyl, C₂-C₈alkenyl, polyethylene glycol, polyethyleneglycolsubstituted C₁-C₆alkenyl, wherein the substituted aryl can be optionallyfurther substituted with halogen, C₁-C₄ alkyl, C₁-C₄haloalkyl, C₁-C₄heteroalkyl, di(C₁-C₂₄alkyl)amino or combinations thereof; R² and R³ arehydrogen or R² and R³ together form a second bond between the carbons towhich they are attached; R⁴ is hydrogen; R⁵ is selected from the groupconsisting of hydrogen and C₁-C₆ alkyl; R⁶ and R⁷ are each independentlyselected from the group consisting of hydrogen, F, Br, Cl and I; and R¹⁰and R¹¹ are each independently selected from the group consisting ofhydrogen, F, Br, Cl, and I.

In some embodiments of Formula (III′), R¹¹ is an aryl substituted withat least one C₁-C₆ alkoxyl and at least one di(C₁-C₂₄alkyl)amino,halogen, C₂-C₈alkenyl, or polyethyleneglycol substituted C₁-C₆alkenyl,wherein the substituted aryl can be optionally further substituted withhalogen, C₁-C₄ alkyl, C₁-C₄haloalkyl, C₁-C₄ heteroalkyl,di(C₁-C₂₄alkyl)amino or combinations thereof; R² and R³ are hydrogen orR² and R³ together form a second bond between the carbons to which theyare attached; R⁴ is hydrogen; R⁵ is selected from the group consistingof hydrogen and C₁-C₆ alkyl; R⁶ and R⁷ are each independently selectedfrom the group consisting of hydrogen, F, Br, Cl and I; and R¹⁰ and R¹¹are each independently selected from the group consisting of hydrogen,F, Br, Cl, and I.

In some embodiments, a compound of Formula (III′) is8-(4-(dimethylamino)-2-ethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(FQI-34), having the structure:

In some embodiments, a compound of Formula (III′) is selected from thegroup consisting of:

In one embodiment, an inhibitor of LSF of formula (I), (II), (III) or(XXVI) is a compound of formula (IV), wherein the formula (IV) has thestructure:

In further embodiments, inhibitors of LSF of formula (I), (II), (III) or(XXVI) can be a compound selected from the group consisting of compoundsof formula (V) to (XXV):

As noted above, compounds any of formula (I) to (XXVI) can inhibit LSF.Without limitations, inhibition of LSF can be determined using anymethod available, including, but not limited to in vitro assays. Forexample, inhibition of LSF can be determined using the methods describedin the Examples section.

In various embodiments, enantiomers, prodrugs, derivatives andpharmaceutically acceptable salts of a compound of any of formula (I) to(XXVI) and (III′) also fall within the scope of the invention.

Yet another aspect of the present invention is directed to the use ofpharmaceutical composition comprising a compound of formula (I) or(III′) as disclosed herein for the manufacturer of a medicament fortreatment of hepatocellular carcinoma. In some embodiments, a compoundof formula (I) can be a compound of any of formula (II) to (XXVI) and(III′). In one embodiment, a compound of formula (IV) is used inpharmaceutical compositions of the invention as disclosed herein. Inadditional embodiments, enantiomers, prodrugs, derivatives andpharmaceutically acceptable salts of a compound of any of formula (III′)or (I) to (XXVI) are also included in the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows exemplary compounds that inhibit cell proliferation intissue culture cells along with the values of IC₅₀ and maximum percentof cell growth inhibition for indicated cell lines. Compounds C1-1 andC1-2 are enantiomers (S- or R-configuration) of compound C1 (FormulaIV), while other compounds are examples of compounds derived fromformula (I), (II) or (III). The designations “R” and “S” as shown incompounds C1-1 and C1-2, respectively, are used to denote the absoluteconfiguration of the molecule about its chiral center. The designations“(+)” and “(−)” as shown in compounds C1-1 and C1-2 are employed todesignate the sign of rotation of plane-polarized light by the compound.

FIGS. 2A-2B shows the structures of LSF inhibitors of structures of FQI1(Formula IV) and FQI2 (Formula V). FIG. 2A shows Factor quinolinoneinhibitor 1 (FQI1) was initially identified as the racemate. The moreactive (5)-enantiomer, (S)-FQI1, and the achiral quinolinone inhibitorFQI2 possess similar chemical properties and biological activities. FIG.2B shows a computationally generated overlay of (S)-FQI1 (cpk) and FQI2(red) using the OpenEye Scientific Software shape similarity comparisonprogram ROCS. Although achiral, FQI2 is capable of adopting similarconformations as (S)-FQI1.

FIGS. 3A-3D show that FQI compounds inhibit LSF transcriptionalactivation and DNA-binding. FIG. 3A shows LSF-dependent fireflyluciferase reporter assays, which were normalized to activity in theabsence of FQIs. USF-dependent reporter assays were performed with FQI1only. Averages with standard deviation (SD) derive from 3-4 independentexperiments. FIG. 3B shows a representative electrophoretic mobilityshift assay (EMSA) of LSF/DNA complexes from in vitro translated LSFincubated with FQI1. “−LSF”: translation extract with no programmed LSF.FIG. 3C shows the fraction of radiolabeled DNA bound to LSF, normalizedto bound levels in absence of FQI1; values were averaged (with standarderror of the mean (SEM)) from 3 EMSAs. FIG. 3D shows a representativechromatin immunoprecipitation assay (ChIP) of myc-LSF-HA binding to theendogenous POLA1 promoter in a human cell line. Myc-LSF-HA expressionwas induced with the small molecule inducer RSL1. In induced cells, FQI1or vehicle was added for the final 12 h of myc-LSF-HA induction.

FIGS. 4A-4C shows in vitro growth inhibition and induction of apoptosisin HCC cells, but not hepatocytes, by FQI compounds. FIG. 4A showsviability of QGY-7703 HCC cells, treated with FQI1 (top) or FQI2(bottom), as assayed by the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cellviability assay. Averages with SEM of three experiments are shown.*P<0.05, by one-tailed t-test. FIG. 4B shows apoptosis in QGY-7703 cellstreated with FQI1, but not with vehicle. The cells were imagedseparately for TUNEL-positivity and DNA at the top. Below: Mergedimages; green for TUNEL (Terminal deoxynucleotidyl transferase dUTP nickend labeling) staining, blue for DAPI (4′,6-diamidino-2-phenylindole)staining. FIG. 4C shows newly plated primary mouse hepatocytes, treatedas indicated, were stained for DNA (blue) and F-actin (green).Binucleate cells are common for adult hepatocytes.

FIGS. 5A-5B shows induction of apoptosis in vitro in aggressive HCCcells (QGY-7703), but not in HepG3 cells, by LSF inhibitors. FIG. 5Ashows the average cell viability (measured by the MTT assay in threeindependent experiments) of HepG3 and QGY-7703 HCC cells treated withincreasing concentrations of FQI1 over a two-day timecourse. Error barsrepresent standard error of the mean. The symbol “*” indicates P<0.05,measured by t-test. FIG. 5B is representative flow cytometric datademonstrating induction of apoptosis in QGY-7703 cells after FQI1treatment. Cells were treated with either vehicle (DMSO) or FQI1 (10 μM)for 48 h; TUNEL assays were performed, followed by flow cytometricanalyses.

FIG. 6 shows that the potent antiproliferative FQIs in NIH 3T3 cellsalso inhibit LSF transcriptional activity. Dual luciferase reporterassays demonstrate the relative degrees of inhibition of LSFtransactivation in NIH 3T3 cells. Compounds were assayed for 0.5, 1.0and 5.0 μM to facilitate comparisons. Shown are averages of threeindependent experiments. Error bars represent SEM.

FIG. 7 shows FQI1-treated cell viability profiles are not affected byexogenous thymidine. QGY-7703 cells were treated with increasingconcentrations of FQI1 over a two-day timecourse, in the presence orabsence of 20 μM thymidine. Shown is a single experiment, performed intriplicate. Error bars represent standard deviation of the replicates.

FIG. 8A-8Q show graphs of the growth inhibition (as % control) versusconcentrations of different FQI (log₁₀) in three different cell lines(NIH 3T3, HeLa, A549) to determine GI₅₀(IC₅₀) values of inhibitors. Alldata are reported in micromolar (μM) concentrations.

FIG. 9A-9K show the results of inhibition of growth of cancer cell linesin vitro by FQI1 (Formula (IV)). The effect of inhibition of cell growth(represented as a fraction of 1) of increasing concentrations of FQI1between 0.2-33 μM is shown. FIG. 9A shows effect of inhibition ofincreasing doses of FQI1 on kidney primary cancer cells (RCC10RGB cellline). FIG. 9B shows effect of inhibition of increasing doses of FQI1 on5 different Hematopoietic cell lines (AMO1, EJM, KMS11, LP1, OCILY10cell lines). FIG. 9C shows effect of increasing doses of FQI1 oninhibition of an Endometrium cancer cell line (HEC151 cell line). FIG.9D shows effect of increasing doses of FQI1 on inhibition of a Breastcancer cell line (MCF7). FIG. 9E shows effect of increasing doses ofFQI1 on inhibition of an upper digestive tract cancer (SCC9) cell line.FIG. 9F shows effect of increasing doses of FQI1 on inhibition of astomach cancer (AGS) cell line. FIG. 9G shows effect of increasing dosesof FQI1 on inhibition of ovary cancer cell lines (A2780, COV362, COV434,EFO27, IGROV1, JHOC5, JHOM1, OAW42, OVCAR4, OVCAR8, OVKATE, RKN, andRMGI cell lines). FIG. 9H shows effect of increasing doses of FQI1 oninhibition of lung cancer primary cell lines (CORL23, NCIH1836,NCIH2073, NCIH2110, NCIH3255, CAL12T, COL0699, CORL51, HCC1171, HCC1195,HCC1359, HCC1833, HCC2108, HCC2935, HCC4006, HCC827, NCUH105, HCIH1694,NCIH1876, NCIH1963, NCIH2023, NCIH2030, NCIH2081, NCIH2141, NCIH2172,NCIH2342, NCIH841, SCLC21H and SQ1 cell lines). FIG. 9I shows effect ofincreasing doses of FQI1 on inhibition on liver cancer cell lines(HEPG2, SNU398 cell lines). FIG. 9J shows effect of increasing doses ofFQI1 on inhibition of large intestine primary cancer cell lines(COL0320, CW2, HCT116, KM12, SNU1033, SNU175, SNU61, SNU81, SNUC5, SW948cell lines). FIG. 9K shows the effect of increasing doses of FQI1 oninhibition all cancer cell lines tested and shown in FIGS. 9A-9J.

FIGS. 10A-10K shows the results of inhibition of growth of cancer celllines in vitro by FQI2 (Formula (V)). The effect of inhibition of cellgrowth (represented as a fraction of 1) of increasing concentrations ofFQI2 between 0.2-33 μM is shown. FIG. 10A shows effect of increasingdoses of FQI2 on inhibition of an upper digestive tract cancer (SCC9)cell line. FIG. 10B shows effect of increasing doses of FQI2 oninhibition of a stomach cancer (AGS) cell line. FIG. 10C shows effect ofincreasing doses of FQI2 on inhibition of ovary cancer cell lines(A2780, COV362, COV434, EFO27, IGROV1, JHOC5, JHOM1, OAW42, OVCAR4,OVCAR8, OVKATE, RKN, and RMGI cell lines). FIG. 10D shows effect ofincreasing doses of FQI2 on inhibition on liver cancer cell lines(HEPG2, SNU398 cell lines). FIG. 10E shows effect of increasing doses ofFQI2 on inhibition of lung cancer primary cell lines (CORL23, NCIH1836,NCIH2073, NCIH2110, NCIH3255, CAL12T, COL0699, CORL51, HCC1171, HCC1195,HCC1359, HCC1833, HCC2108, HCC2935, HCC4006, HCC827, NCUH105, HCIH1694,NCIH1876, NCIH1963, NCIH2023, NCIH2030, NCIH2081, NCIH2141, NCIH2172,NCIH2342, NCIH841, SCLC21H and SQ1 cell lines). FIG. 10F shows effect ofincreasing doses of FQI2 on inhibition of large intestine primary cancercell lines (COL0320, CW2, HCT116, KM12, SNU1033, SNU175, SNU61, SNU81,SNUC5, SW948 cell lines). FIG. 10G shows effect of inhibition ofincreasing doses of FQI2 on kidney primary cancer cells (RCC10RGB cellline). FIG. 10H shows effect of inhibition of increasing doses of FQI2on 5 different Hematopoietic cell lines (AMO1, EJM, KMS11, LP1, OCILY10cell lines). FIG. 10I shows effect of increasing doses of FQI2 oninhibition of an Endometrium cancer cell line (HEC151 cell line). FIG.10J shows effect of increasing doses of FQI2 on inhibition of a Breastcancer cell line (MCF7). FIG. 10K shows the effect of increasing dosesof FQI2 on inhibition of all cancer cell lines tested and shown in FIGS.10A-10J.

FIGS. 11A-11C show growth inhibition (as % control) versus FQI-34concentration.

FIG. 12 shows LSF-dependent firefly luciferase reporter activity inNIH-3T3 cells from transfection using an LSF expression construct, andan internal control.

FIGS. 13A and 13B show target binding assessment by Cellular ThermalShift Assay (CETSA). FIG. 13A shows representative western blot forCETSA analysis. FIG. 13B shows quantification of CETSA western blots(N≥3), mean±S.E.M. Mean AUC for each graph is shown. ***p<0.001,**p<0.01, NS p>0.05.

FIG. 14 shows mean absorbance readings at 620 nm±S.D for air-exposed anddry FQI-34. Dashed line at Y=0.01 represents the precipitation point.

FIGS. 15A and 15B show average plasma concentration (ng/mL) vs time(hour) for IV dosing (FIG. 15A) and for PO dosing (FIG. 15B). Insetsshow individual rat values.

FIGS. 16A and 16B show target binding assessment by Cellular ThermalShift Assay for compounds FQI-34, FQI-Br and FQI-Cl. FIG. 16A showsrepresentative western blots against LSF (N≥3). FIG. 16B showsquantification of anti-LSF western blots (N≥3). Area-under-the-curve(AUC) was calculated. Mean AUC+SEM is plotted, with mean DMSO AUCindicated as dashed line. Significance determined using GraphPad PRISMunpaired, parametric t test. *<0.05**<0.01***<0.001****<0.0001.

DETAILED DESCRIPTION OF THE INVENTION

In some embodiments, the present invention is based upon the discoverythat expression of LSF is increased in subjects, e.g. human subjectswith HCC. The inventors have also discovered small-molecule compoundsthat inhibit LSF. The inventors have discovered that the small moleculeinhibitors of LSF as disclosed herein can cause cell death of cancercell lines and primary cancer cells in an in vitro assay, e.g., HCCcancer cell lines, breast cancer cell lines, colon cancer cell lines,ovarian cancer cell lines etc., Studies performed by the inventors incollaboration with other scientists revealed that the small moleculeinhibitors as disclosed herein can decrease tumorigenesis and metastasisof HCC in an in vivo mouse model of HCC. Therefore, the small-moleculeinhibitors of LSF disclosed herein can be used in a method for treatmentof cancers in subjects, e.g. HCC and other cancers human subjects.

Accordingly, in some embodiments, the present invention provides methodsand compositions for inhibition of LSF and can be used for treatment ofhepatocellular carcinoma in a subject. In some embodiments, the presentinvention relates in part to the use of small-molecule compounds toinhibit LSF for treatment of cancer, e.g. hepatocellular carcinoma(HCC), liver cancers, breast cancers, ovarian cancers, colon cancers,cervical cancers, melanomas etc. . . . .

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

Definitions for Chemical Functional Groups

The structure definitions such as “alkyl” are provided below fornomenclature purposes. They do not exclude the meaning as those acquiredin the art to which this invention pertains. The term “alkyl” as usedherein refers to a linear, branched, or cyclic saturated hydrocarbongroup typically although not necessarily containing 1 to about 24 carbonatoms, preferably 1 to about 12 carbon atoms, more preferably 1 to about6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like, as well ascycloalkyl groups such as cyclopentyl, cyclohexyl and the like. The term“cycloalkyl” intends a cyclic alkyl group, typically having 4 to 10,preferably 5 to 8, carbon atoms. The term “substituted alkyl” refers toalkyl substituted with one or more substituent groups, and the terms“heteroalkyl” and “heteroatom-containing alkyl” refer to alkyl in whichat least one carbon atom is replaced with a heteroatom. The term“haloalkyl” as used herein refers to an alkyl structure with at leastone substituent of fluorine, chorine, bromine or iodine, or withcombinations thereof. If not otherwise indicated, the term “alkyl”includes linear, branched, cyclic, unsubstituted, substituted, and/orheteroatom-containing alkyl, respectively.

The term “alkenyl” as used herein refers to a linear, branched, orcyclic hydrocarbon group of 2 to about 16 carbon atoms containing atleast one double bond, such as ethenyl, n-propenyl, isopropenyl,n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl,eicosenyl, tetracosenyl, and the like. Preferred alkenyl groups hereincontain 2 to about 8 carbon atoms. The term “substituted alkenyl” refersto alkenyl substituted with one or more substituent groups, and theterms “heteroatom-containing alkenyl” and “heteroalkenyl” refer toalkenyl in which at least one carbon atom is replaced with a heteroatom.If not otherwise indicated, the term “alkenyl” includes linear,branched, cyclic, unsubstituted, substituted, and/orheteroatom-containing alkenyl, respectively.

The term “alkynyl” as used herein refers to a linear or branched-chainhydrocarbon group having one or more carbon-carbon triple-bonds andhaving from 2 to about 8 carbon atoms. Examples of alkynyl groupsinclude ethynyl, propynyl, butynyl and the like.

The term “alkoxy” as used herein intends an alkyl group bound through asingle, terminal ether linkage; that is, an “alkoxy” group may berepresented as —O-alkyl where alkyl is as defined herein.

The term “aryl” as used herein, and unless otherwise specified, refersto an aromatic substituent containing a single aromatic ring or multiplearomatic rings that are fused together, directly linked, or indirectlylinked (such that the different aromatic rings are bound to a commongroup such as a methylene or ethylene moiety). Preferred aryl groupscontain 5 to 24 carbon atoms, and particularly preferred aryl groupscontain 5 to 14 carbon atoms. Exemplary aryl groups contain one aromaticring or two fused or linked aromatic rings, e.g., phenyl, naphthyl,biphenyl, diphenylether, diphenylamine, benzophenone, and the like.“Substituted aryl” refers to an aryl moiety substituted with one or moresubstituent groups, and the terms “heteroatom-containing aryl” and“heteroaryl” refer to aryl substituents in which at least one carbonatom is replaced with a heteroatom such as oxygen, nitrogen and sulfur.The term “heteroaryl” includes ring systems such as pyridine, quinoline,furan, thiophene, pyrrole, imidazole and pyrazole.

The term “heterocycyl” as used herein refers to a single ring ormultiple rings that are fused together, directly linked, or indirectlyinked (such that the different rings are bound to a common group such asa methylene or ethylene moiety), in which at least one carbon atom isreplaced with a heteroatom such as oxygen, nitrogen and sulfur.Preferred heterocycyl groups contain 5 to 24 carbon atoms, andparticularly preferred heterocycyl groups contain 5 to 14 carbon atoms.For example, a heterocycyl group can be a five-membered ring with atleast one carbon replaced by oxygen or nitrogen.

The terms “cyclic” and “ring” refer to alicyclic or aromatic groups thatmay or may not be substituted and/or heteroatom containing, and that maybe monocyclic, bicyclic, or polycyclic. The term “alicyclic” is used inthe conventional sense to refer to an aliphatic cyclic moiety, asopposed to an aromatic cyclic moiety, and may be monocyclic, bicyclic orpolycyclic. In one embodiment, the bicyclic or polycyclic ring may befused ring. The fusion of the ring may be across a bond between twoatoms, i.e. two cyclic rings share one bond or two atoms, for example, adecalin; the fusion of the ring may be across a sequence of atoms, i.e.two cyclic rings share three or more atoms, for example a norbornane.

By “substituted” as in “substituted alkyl,” “substituted aryl,” and thelike, as alluded to in some of the definitions as described herein, ismeant that in the alkyl, aryl, or other moiety, at least one hydrogenatom bound to a carbon (or other) atom is replaced with one or morenon-hydrogen substituents. Examples of such substituents include,without limitation: halogen, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄alkynyloxy, C₅-C₂₄ aryloxy, C₆-C₂₄ aryl alkoxy, C₆-C₂₄ alkyl aryloxy,acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₄ arylcarbonyl(—CO-aryl)), acyloxy (—O-acyl, including C₂-C₂₄ alkylcarbonyloxy(—O—CO-alkyl) and C₆-C₂₄ arylcarbonyloxy (—O—CO-aryl)), C₂-C₂₄alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₄ aryloxycarbonyl (—(CO)—O-aryl),halocarbonyl (—CO)—X where X is halo), C₂-C₂₄ alkylcarbonato(—O—(CO)—O-alkyl), C₆-C₂₄ arylcarbonato (—O—(CO)—O-aryl), carboxy(—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂), mono-(C₁-C₂₄alkyl)-substituted carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), di-(C₁-C₂₄alkyl)-substituted carbamoyl (—(CO)—N(C₁-C₂₄ alkyl)₂), mono-(C₅-C₂₄aryl)-substituted carbamoyl (—(CO)—NH-aryl), di-(C₅-C₂₄aryl)-substituted carbamoyl (—(CO)—N(C₅-C₂₄ aryl)₂), di-N—(C₁-C₂₄alkyl), N—(C₅-C₂₄ aryl)-substituted carbamoyl, thiocarbamoyl(—(CS)—NH₂), mono-(C₁-C₂₄ alkyl)-substituted thiocarbamoyl(—(CO)—NH(C₁-C₂₄ alkyl)), di-(C₁-C₂₄ alkyl)-substituted thiocarbamoyl(—(CO)—N(C₁-C₂₄ alkyl)₂), mono-(C₆-C₂₄ aryl)-substituted thiocarbamoyl(—(CO)—NH-aryl), di-(C₆-C₂₄ aryl)-substituted thiocarbamoyl(—(CO)—N(C₅-C₂₄ aryl)₂), di-N—(C₁-C₂₄ alkyl),N—(C₆-C₂₄ aryl)-substitutedthiocarbamoyl, carbamido (—NH—(CO)—NH₂), cyano(—C≡N), cyanato (—O—C≡N),thiocyanato (—S—CEN), formyl (—(CO)—H), thioformyl (—(CS)—H), amino(—NH₂), mono-(C₁-C₂₄ alkyl)-substituted amino, di-(C₁-C₂₄alkyl)-substituted amino, mono-(C₆-C₂₄ aryl)-substituted amino,di-(C₆-C₂₄ aryl)-substituted amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl),C₆-C₂₄ arylamido (—NH—(CO)-aryl), imino (—CR═NH where R=hydrogen, C₁-C₂₄alkyl, C₅-C₂₄ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), C₂-C₂₀alkylimino (—CR═N(alkyl), where R=hydrogen, C₁-C₂₄ alkyl, C₅-C₂₄ aryl,C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), arylimino (—CR═N(aryl), whereR=hydrogen, C₁-C₂₀ alkyl, C₅-C₂₄ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl,etc.), nitro (—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato(—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl; also termed “alkylthio”),C₅-C₂₄ arylsulfanyl (—S-aryl; also termed “arylthio”), C₁-C₂₄alkylsulfinyl (—(SO)-alkyl), C₅-C₂₄ arylsulfinyl (—(SO)-aryl), C₁-C₂₄alkylsulfonyl (—SO₂-alkyl), C₅-C₂₄ arylsulfonyl (—SO₂-aryl), boryl(—BH₂), borono (—B(OH)₂), boronato (—B(OR)₂ where R is alkyl or otherhydrocarbyl), phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂),phosphinato (—P(O)(O⁻), phospho (—PO₂), phosphino (—PH₂); and thehydrocarbyl moieties C₁-C₂₄ alkyl (preferably C₁-C₁₂ alkyl, morepreferably C₁-C₆ alkyl), C₂-C₂₄ alkenyl (preferably C₂-C₁₂ alkenyl, morepreferably C₂-C₆ alkenyl), C₂-C₂₄ alkynyl (preferably C₂-C₁₂ alkynyl,more preferably C₂-C₆ alkynyl), and C₅-C₂₄ aryl (preferably C₅-C₁₄aryl). In preferred embodiments, the substituents as used herein arehalogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ heteroalkyl or C₁-C₆alkoxy.

In addition, the functional groups as described herein may, if aparticular group permits, be further substituted with one or moreadditional functional groups or with one or more hydrocarbyl moietiessuch as those specifically enumerated herein. Analogously, thehydrocarbyl moieties described herein may be further substituted withone or more functional groups or additional hydrocarbyl moieties such asthose specifically enumerated.

Other Definitions

The term “inhibit LSF” as used herein refers to inhibiting expression(level) of LSF and/or biological activity of LSF. In some embodiments,the term “inhibit LSF” refers to a decrease in the protein level of LSFand/or gene transcript level of LSF. For example, inhibition of LSF canresult in a reduction in the gene expression of TFCP2 encoding LSF. Theterm “inhibit LSF” also refers to a down-regulation or an inhibition ofbiological activity of LSF, e.g. the function of LSF to modulateexpression of LSF-regulated downstream genes such as; thymidylatesynthetase (TYMS), secreted phosphoprotein 1 (SPP1), complement factor H(CFH) and fibronectin 1 (FN1) (see Porta-de-la-Riva M, et al (2011) J.Biochem. 435:563-8, which is incorporated herein in its entirety byreference).

The terms “cellular LSF activity” and “biological activity of LSF” areused herein interchangeably. Both terms refer to the ability of LSF toregulate cellular processes downstream of LSF, for example, to modulatethe expression of genes that are downstream of LSF. In some embodiments,the biological activity of LSF can elicit a stimulatory effect onexpression of LSF-downstream genes. In other embodiments, the biologicalactivity of LSF can induce an inhibitory effect on expression ofLSF-downstream genes. In yet other embodiments, the biological activityof LSF may be due to interactions with other cellular proteins.

The phrase “level of LSF” as used herein encompasses the expressionand/or biological activity of LSF. As described herein, the term“expression” refers to the amount of the protein obtained by translationof RNA transcribed from a gene, and/or the amount of RNA transcribedfrom a gene.

The term “regulate” used herein in reference to expression of a gene,refers to producing an effect on, for example, gene expression. In someembodiments, the effect can be stimulatory, such as increasingexpression of a gene. In some embodiments, the effect can be inhibitory,such as decreasing expression of a gene. The terms “regulate” and“modulate” are interchangeably used herein.

The terms “inhibitors of LSF” and “LSF inhibitors” used interchangeablyherein, generally refers to agents that inhibit LSF. They can be ofsynthetic or biological origins. They can be organic, or inorganicmolecules, or peptides, antibodies or antisense RNA that inhibit LSF.Inhibitors of LSF of the invention are chemical entities or moleculesthat can inhibit expression of LSF and/or biological activity of LSF, asdisclosed herein, for example, compounds of any of formula (I) to(XXVI), and enantiomers, prodrugs, derivatives and pharmaceuticallyacceptable salts thereof, which are discussed further in the section

Inhibitors of LSF.

The ability of a compound to inhibit LSF can be assessed by measuring adecrease in expression of LSF as compared to the level of LSF in theabsence of inhibitors of LSF. In some embodiments, the ability of acompound to inhibit LSF can be assessed by measuring a decrease in thebiological activity, e.g., transcriptional activity of LSF as comparedto the level of transcriptional activity of LSF in the absence ofinhibitors of LSF. The expression of LSF includes the amount of RNAtranscribed from a gene, e.g. TFCP2 that encodes LSF, and/or the amountof LSF proteins that is obtained by translation of RNA transcribed froma gene, e.g. TFCP2. For example, a LSF inhibitor as disclosed herein caninhibit expression of LSF by at least about 10%, at least about 15%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, 95%, 99% or even 100%, as compared to a reference levelin the absence of a LSF inhibitor.

Additionally, ability of a compound to inhibit LSF can be also assessedby measuring a decrease in or an inhibition of biological activity ofLSF as compared to a negative control, e.g. the experimental conditionin the absence of LSF inhibitors. The biological activity of LSF canrefer to the ability of LSF to modulate expression of LSF-targeted genessuch as thymidylate synthase (TYMS) and/or LSF-downstream genes, such assecreted phosphoprotein 1 (SPP1), complement factor H (CFH) and othertumor-associated genes (see Yoo et al., PNAS, 2010, 107; 8357-8362,which is incorporated herein in its entirety by reference). Accordingly,a LSF inhibitor as disclosed herein can inhibit biological activity ofLSF, such as a decrease in expression of SPP1 that encodes OPN (alsoknown as secreted phosphoprotein 1, SPP1), by at least about 10%, atleast about 15%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, 95%, 99% or even 100%, as comparedto a reference level in the absence of a LSF inhibitor. In someembodiments, ability of a compound to inhibit LSF is assessed byinhibition of LSF-induced tumorigenesis and metastasis of cancer cells,e.g. hepatocellular carcinoma cells in vitro or in an in vivo animalmodel as demonstrated in the Examples herein, as compared to a referencecondition without treatment with such a LSF inhibitor. In suchembodiments, a LSF inhibitor can decrease a tumor weight and volume byat least about 15%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, 95%, 99% or even 100%, ascompared to no treatment with a LSF inhibitor.

The term ‘disorder’ or ‘disease’ used interchangeably herein, refers toany alteration in the state of the body or of some of its organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with the person. Adisease or disorder can also relate to distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, indisposition,affection. In one embodiment, the disorder or disease is cancer. In oneembodiment, the disease or disorder is liver cancer, e.g.,hepatocellular carcinoma. In one embodiment, the disease or disorder isa cancer selected from the group selected from: colon cancer, breastcancer, ovarian cancer, melanomia, endometrium cancer, pancreaticcancer, prostate cancer, bone cancer, kidney cancer, leukemia, largeintestine cancer, lung cancer, small cell lung carcinoma (SSLC), stomachcancer and other cancers.

The term ‘cancer’ and ‘malignancy’ are used interchangeably herein, andrefer to a disease that is characterized by uncontrolled, abnormalgrowth of cells. Cancer cells can spread locally or through thebloodstream and lymphatic system to other parts of the body. The term isalso intended to include any disease of an organ or tissue in mammalscharacterized by poorly controlled or uncontrolled multiplication ofnormal or abnormal cells in that tissue and its effect on the body as awhole. Cancer diseases within the scope of the definition comprisebenign neoplasms, dysplasias, hyperplasias as well as neoplasms showingmetastatic growth or any other transformations like e.g. leukoplakiaswhich often precede a breakout of cancer. The term cancer also includesmetastases which are cancer cells (e.g. a primary tumor, or a metastasistumor) which has migrated to other locations in the subject and toestablish new tumors at such locations. A small molecule LSF inhibitoras disclosed herein which “inhibits” cancer metastasis may result in thedelayed appearance of secondary tumors, slowed development of primary orsecondary tumors, decreased occurrence of secondary tumors, slowed ordecreased severity of secondary effects of disease, arrested tumorgrowth and regression of tumors, among others. In the extreme, completeinhibition is referred to herein as prevention (e.g., virtually completeinhibition, no metastasis if it had not occurred, no further metastasisif there had already been metastasis of a cancer, or virtually completeinhibition of the growth of a primary tumor caused by re-seeding of thetumor by a metastasized cell.

A “cancer cell” refers to a cancerous, pre-cancerous or transformedcell, either in vivo, ex vivo, and in tissue culture, that hasspontaneous or induced phenotypic changes that do not necessarilyinvolve the uptake of new genetic material. Although transformation canarise from infection with a transforming virus and incorporation of newgenomic nucleic acid, or uptake of exogenous nucleic acid, it can alsoarise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation/cancer is associated with,e.g., morphological changes, immortalization of cells, aberrant growthcontrol, foci formation, anchorage dependence, proliferation,malignancy, contact inhibition and density limitation of growth, growthfactor or serum dependence, tumor specific markers levels, invasiveness,tumor growth or suppression in suitable animal hosts such as nude mice,and the like, in vitro, in vivo, and ex vivo (see also Freshney, Cultureof Animal Cells: A Manual of Basic Technique (3rd ed. 1994)).

A “tumorigenic cell,” as used herein, is a cell that, when introducedinto a suitable site in a subject, can form a tumor. The cell may benon-metastatic or metastatic. A variety of types of tumorigenic and/ormetastatic cells can be used in a method of the invention, includingcells from metastatic epithelial cancers, carcinomas, melanoma,leukemia, etc. The tumor cells may be, e.g., from cancers of breast,lung, colon, bladder, prostate, liver, gastrointestinal tract,endometrium, tracheal-bronchial tract, pancreas, liver, uterus, ovary,nasopharynges, prostate, bone or bone marrow, brain, skin or othersuitable tissues or organs. In a preferred embodiment, the cancer cellsare of human origin.

The term “tumor” or “tumor cell” are used interchangeably herein, refersto the tissue mass or tissue type of cell that is undergoing abnormalproliferation.

A “metastatic” cell, as used herein, refers to a cell that has apotential for metastasis and, when used in a method of the invention, isable to seed a tumor or a cell colony of interest. A “highly metastatic”cell, as used herein, refers to a cell that has a high potential formetastasis; e.g., cells from a cell line such as, but not limited toLM2, MDA-MB-231, PC-3, DU-145, Lewis Lung carcinoma, as describedherein, can be considered to be highly metastatic cells. Metastaticcells can be generated in a variety of ways, which are discussed furtherbelow.

A “sarcoma” refers to a type of cancer cell that is derived fromconnective tissue, e.g., bone (osteosarcoma) cartilage (chondrosarcoma),muscle (rhabdomyosarcoma or rhabdosarcoma), fat cells (liposarcoma),lymphoid tissue (lymphosarcoma), collagen-producing fibroblasts(fibrosarcoma). Sarcomas may be induced by infection with certainviruses, e.g., Kaposi's sarcoma, Rous sarcoma virus, etc.

The term “tissue” is intended to include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, muscles, smoothmuscles, and organs.

The term “subject” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment. The term “subject”and “individual” are used interchangeably herein, and refer to ananimal, for example a human, to whom treatment, including prophylactictreatment, with the cells according to the present invention, isprovided. The “non-human animals” of the invention include mammals suchas rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-humanprimates.

The terms “a reference sample” or “a reference level” as usedinterchangeably herein refer to a negative control of the condition. Forexample, in the context of treatment, a reference level is the level ifa subject is not treated. In some embodiments, a reference level in thecontext of diagnosis is the level present in a normal healthy subject.The term “normal healthy subject” refers to a subject who has nosymptoms of any diseases or disorders, or who is not identified with anydiseases or disorders, or who is not on any medication treatment, or asubject who is identified as healthy by physicians based on medicalexaminations. In some embodiments, a reference level or sample usedherein refers to the level measured at a previous time point from asubject being treated.

The term “tissue” is intended to include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, muscles, smoothmuscles, and organs. In one embodiment, the tissue is liver tissue.

The terms “treat”, “treatment” and “treating” used interchangeably, withrespect to treatment of a disease or disorder, mean preventing thedevelopment of the disease, or altering the course of the disease (forexample, but not limited to, slowing the progression of the disease), orreversing a symptom of the disease or reducing one or more symptomsand/or one or more biochemical markers in a subject, preventing one ormore symptoms from worsening or progressing, promoting recovery orimproving prognosis in a subject who is at risk of the disease, as wellas slowing or reducing progression of existing disease. The termtreating encompasses reducing or alleviating at least one adverse effector symptom of a condition, disease or disorder associated withinappropriate proliferation, for example cancer. As used herein withrespect to cancer, the term treating is used to refer to the reductionof a symptom and/or a biochemical marker of in appropriateproliferation, for example a reduction in at least one biochemicalmarker of cancer by at least 10%. For example but are not limited to, areduction in a biochemical marker of cancer, for example a reduction in,as an illustrative example only, at least one of the followingbiomarkers; CD44, telomerase, TGF-α, TGF-β, erbB-2, erbB-3, MUC1, MUC2,CK20, PSA, CA125, FOBT, by 10%, or a reduction in the rate ofproliferation of the cancer cells by 10%, would be considered effectivetreatments by the methods as disclosed herein. As alternative examples,a reduction in a symptom of cancer, for example, a slowing of the rateof growth of the cancer by 10% or a cessation of the increase in tumorsize, or a reduction in the size of a tumor by 10% or a reduction in thetumor spread (i.e. tumor metastasis) by 10% would also be considered asan effective treatments by the methods as disclosed herein. In otherembodiments, treatment can be therapeutic in terms of eliminating orreducing at least one symptom of the condition or disease. For example,in the case of HCC, therapeutic treatment refers to inhibiting ordelaying the progression of HCC in a subject that is already inflictedwith HCC. Measurable lessening includes any statistically significantdecline in a measurable marker or symptom, such as measuring a tumorsize or level of a biomarker.

As used herein, the term “treating” includes preventing the progressionand/or reducing or reversing at least one adverse effect or symptom of acondition, disease or disorder associated with inappropriateproliferation, for example cancer. Accordingly, in some embodiments,treatment can be prophylactic in terms of completely or partiallypreventing a disease or sign or symptom of cancer. For example, subjectsat high risk of cancer, e.g., HCC, such as HBV or HCV, can be subjectedto prophylactic treatment to prevent the onset of HCC. In someembodiments, prophylactic treatment can be administered to subjects whohad prior treatment of a disease and the disease is in remission. Forexample, for subjects who have their HCC tumors removed or stabilized byprevious therapeutic methods can be prophylactically treated (e.g. witha LSF inhibitor as disclosed herein) to prevent the recurrence andmetastasis of HCC.

As used herein, the terms “prevent,” “preventing” and “prevention” referto the avoidance or delay in manifestation of one or more symptoms ormeasurable markers of a disease or disorder. A delay in themanifestation of a symptom or marker is a delay relative to the time atwhich such symptom or marker manifests in a control or untreated subjectwith a similar likelihood or susceptibility of developing the disease ordisorder. The terms “prevent,” “preventing” and “prevention” include notonly the complete avoidance or prevention of symptoms or markers, butalso a reduced severity or degree of any one of those symptoms ormarkers, relative to those symptoms or markers arising in a control ornon-treated individual with a similar likelihood or susceptibility ofdeveloping the disease or disorder, or relative to symptoms or markerslikely to arise based on historical or statistical measures ofpopulations affected by the disease or disorder. By “reduced severity”is meant at least a 10% reduction in the severity or degree of a symptomor measurable disease marker, relative to a control or reference, e.g.,at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or even100% (i.e., no symptoms or measurable markers).

The terms “up-regulate”, “increase” or “activate” are all used herein togenerally mean an increase by a statically significant amount; for theavoidance of any doubt, the terms “up-regulate”, “increase” or “higher”means an increase of at least 10% as compared to a reference level, forexample an increase of at least about 20%, or at least about 30%, or atleast about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or a 100%increase or more, or any increase between 10-100% as compared to areference level, or an increase greater than 100%, for example, anincrease at least about a 2-fold, or at least about a 3-fold, or atleast about a 4-fold, or at least about a 5-fold or at least about a10-fold increase, or any increase between 2-fold and 10-fold or greateras compared to a reference level. When “increase” is used in the contextof the expression or activity of a gene or protein, it refers to apositive change in protein or nucleic acid level or activity in a cell,a cell extract, or a cell supernatant. For example, such an increase maybe due to increased RNA stability, transcription, or translation, ordecreased protein degradation. Preferably, this increase is at least 5%,at least about 10%, at least about 25%, at least about 50%, at leastabout 75%, at least about 80%, at least about 100%, at least about 200%,or even about 500% or more over the level of expression or activityunder control conditions.

The terms “lower”, “reduced”, “reduction” or “decrease”, “down-regulate”or “inhibit” are all used herein generally to mean a decrease by astatistically significant amount. However, for avoidance of doubt,“lower”, “reduced”, “reduction” or “decrease” or “inhibit” means adecrease by at least 10% as compared to a reference level, for example adecrease by at least about 20%, or at least about 30%, or at least about40%, or at least about 50%, or at least about 60%, or at least about70%, or at least about 80%, or at least about 90% or up to and includinga 100% decrease (i.e. absent level as compared to a reference sample),or any decrease between 10-100% as compared to a reference level. When“decrease” or “inhibition” is used in the context of the level ofexpression or activity of a gene or a protein, e.g. LSF, it refers to areduction in protein or nucleic acid level or activity in a cell, a cellextract, or a cell supernatant. For example, such a decrease may be dueto reduced RNA stability, transcription, or translation, increasedprotein degradation, or RNA interference. In some embodiments, thesmall-molecule LSF inhibitors as disclosed herein decrease the activityor expression of LSF. Preferably, this decrease is at least about 5%, atleast about 10%, at least about 25%, at least about 50%, at least about75%, at least about 80%, or even at least about 90% of the level ofexpression or activity under control conditions. The term “level” asused herein in reference to LSF refers to expression or activity of LSF.

The terms “significantly different than,”, “statistically significant,”and similar phrases refer to comparisons between data or othermeasurements, wherein the differences between two compared individualsor groups are evidently or reasonably different to the trained observer,or statistically significant (if the phrase includes the term“statistically” or if there is some indication of statistical test, suchas a p-value, or if the data, when analyzed, produce a statisticaldifference by standard statistical tests known in the art).

A “pharmaceutical composition” refers to a chemical or biologicalcomposition suitable for administration to a mammalian subject. Suchcompositions may be specifically formulated for administration via oneor more of a number of routes, including but not limited to, oral,parenteral, intravenous, intraarterial, subcutaneous, intranasal,sublingual, intraspinal, intracerebroventricular, and the like.

The term “effective amount” as used herein refers to the amount of atleast one agent of pharmaceutical composition to reduce or stop at leastone symptom of the abnormal proliferation, for example a symptom of acancer or malignancy. For example, an effective amount using the methodsas disclosed herein would be considered as the amount sufficient toreduce a symptom of the abnormal proliferation, for example at least onesymptom of a cancer or malignancy by at least 10%. An effective amountas used herein would also include an amount sufficient to prevent ordelay the development of a symptom of the disease, alter the course of asymptom disease (for example but not limited to, slow the progression ofa symptom of the disease), or reverse a symptom of the disease.Accordingly, the term “effective amount” or “therapeutically effectiveamount” as used herein refers to the amount of therapeutic agent (e.g.at least one small molecule inhibitor of LSF of Formula (I) to (XXVI) asdisclosed herein) of pharmaceutical composition to alleviate at leastsome of the symptoms of cancer e.g. HCC. Stated another way,“therapeutically effective amount” of a small molecule LSF inhibitor asdisclosed herein is the amount of a LSF inhibitor which exerts abeneficial effect on, for example, cancer, e.g., HCC. Beneficial effectsinclude inhibition or delay of cancer, e.g., HCC progression. The dosageadministered, as single or multiple doses, to an individual will varydepending upon a variety of factors, including pharmacokineticproperties of a LSF inhibitor, the route of administration, conditionsand characteristics (sex, age, body weight, health, size) of subjects,extent of symptoms, concurrent treatments, frequency of treatment andthe effect desired.

An “agent” is a chemical molecule of synthetic or biological origin. Inthe context of the present invention, an agent is generally a moleculethat can be used in a pharmaceutical composition. In some embodiments,the agent is chemotherapeutic agents. In some embodiments, the agent issmall-molecule LSF inhibitors as disclosed herein. In some embodiments,the agent can provide a therapeutic value. In some embodiments, thesmall molecule LSF inhibitors as disclosed herein can be used as apreventative or prophylactic treatment for prevention of cancer, e.g.,where a subject is at risk or is likely to develop cancer. In otherembodiments, the agent is used solely to implement the invention, e.g.pharmaceutically acceptable carriers as disclosed herein.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation, for example the carrierdoes not decrease the impact of the agent on the treatment. In otherwords, a carrier is pharmaceutically inert. The terms “physiologicallytolerable carriers” and “biocompatible delivery vehicles” are usedinterchangeably.

The terms “administered” and “subjected” are used interchangeably in thecontext of treatment of a disease or disorder. Both terms refer to asubject being treated with an effective dose of pharmaceuticalcomposition comprising a LSF inhibitor of the invention by methods ofadministration such as parenteral or systemic administration.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection, infusion and other injection orinfusion techniques, without limitation. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration of a pharmaceutical composition comprising at least aninhibitor of LSF as disclosed herein such that it enters the animal'ssystem and, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%. The present invention is further explained in detail by thefollowing, including the Examples, but the scope of the invention shouldnot be limited thereto.

Latent SV40 Factor (LSF)

LSF is short for late SV40 factor or late Simian Virus 40 factor, and isalso known as aliases LBP-1 (leader binding protein-1), LBP-1c, LBP-1d,UBP-1 (upstream binding protein-1), SEF (SAA3 enhancer factor), TFCP2(transcription factor CP2) and CP2. LSF regulates diverse cellular andviral promoters (7, 8).

For the purposes of this disclosure, the term “LSF” refers to atranscription factor encoded by the gene TFCP2. The sequences of nucleicacid and amino acid of LSF are known and have been assigned NCBIaccession numbers for different species such as human, mouse and rat. Inparticular, the NCBI accession numbers for the nucleic acid and aminoacid sequence of LSF for human are NM_005653 (SEQ ID NO: 1) andNP_005644 (SEQ ID NO: 2), respectively.

LSF is a DNA-binding transcription factor that is required in multiplecell types for cell cycle progression. It binds to the alpha-globinpromoter and activates transcription of the alpha-globin gene. It hasbeen reported that LSF facilitates entry into G1/S phase of the cellcycle, promotes DNA synthesis, and functions as an antiapoptotic factor(9).

LSF also regulates erythroid gene expression, plays a role in thetranscriptional switch of globin gene promoters, and it activates manyother cellular and viral gene promoters. The gene product interacts withcertain inflammatory response factors, and polymorphisms of this genecan be involved in the pathogenesis of Alzheimer's disease.

A major cellular target of LSF is the thymidylate synthase (TS) gene(TYMS), which encodes the rate-limiting enzyme in the production ofdTTP, required for DNA synthesis (9). TS has been a long-standingchemotherapeutic target for cancer treatments, and recently it wasdiscussed that elevated levels of LSF in hepatocellular carcinoma celllines can contribute to chemoresistance to one commonly utilizedthymidylate synthetase inhibitor, 5-fluorouracil. Inhibition of LSFabrogates TS induction, induces either arrest at the G1/S transition, orin apoptosis after entry into S phase. Thus, LSF plays an important rolein DNA synthesis and cell survival.

In the liver, LSF is activated by inflammatory cytokines and regulatesthe expression of acute phase proteins (10, 11).

Inhibitors of LSF

The present invention relates in part to methods and compositions toinhibit LSF, more specifically, with small-molecule LSF inhibitors. Insome embodiments, inhibitors of LSF as disclosed herein can be used toinhibit the cellular LSF activity. In some embodiments, LSF inhibitorsas disclosed herein can decrease expression (level) of LSF.

One of the major targets of LSF is thymidylate synthase (TS) gene(TYMS), which encodes the rate-limiting enzyme in the production ofdTTP, required for DNA synthesis (9). Additional examples ofLSF-downstream genes are disclosed in Yoo et al, PNAS, 2010, 107;8357-8362, and include without limitation SPP1 (encoding osetopontin),complement factor H (CFH), TSPAN8, S100A10, CDH17, EFNB2, ZEB1, REG1A,REG3A, SAA4, TAGLN, FGFR2, EGFR, CYP2B7P1, CYP2B6, GPX2, DPYD, PKLR,LEF1, ICAM2 and IGFBP7.

In some embodiments, the genes downstream of LSF are tumor-associatedgenes, such as relating to invasion and metastasis, angiogenesis,epithelial-mesenchymal transition (EMT), cell growth, drug metabolism,senescence, cell adhesion, glycolysis, Wnt signaling, growth andregeneration, inflammatory response, e.g. acute phase proteins, andmodulators of matrix-degrading enzymes e.g. MMP9. In variousembodiments, LSF is a transcription factor encoded by TFCP2. Thus,inhibiting LSF can disrupt or inhibit LSF binding to DNA and/orinteraction of LSF with other proteins to form a complex.

Accordingly, in some embodiments, inhibition of cellular LSF activitycan be determined by measuring the level of downstream genes regulatedby the transcription factor LSF. The effect of LSF on expression (level)of LSF-targeted or LSF-downstream genes can be stimulatory orinhibitory. For example, one gene induced by LSF is SPP1 encoding OPN.Thus, an inhibition of biological activity of LSF results in a decreasein level of SPP1 mRNA and/or a decrease in the amount of the respectiveencoded protein, OPN. In another embodiment, one gene inhibited by LSFis TAGLN. Thus, an inhibition of biological activity of LSF leads to anincrease in level of TAGLN mRNA. In some embodiments, the cellularactivity of LSF can be measured by a reduction in the level of TS.

In further embodiments, inhibition of LSF can decrease expression ofLSF, for example, a reduction in protein level, and/or a decrease ingene transcript level (e.g. mRNA) of LSF.

One aspect of the present invention provides methods of identifyinginhibitors of LSF, for example, by the method comprising measuring theLSF activity in a LSF-dependent luciferase reporter assay as describedin Example 1. As disclosed herein, inhibitors of LSF can decreasefunctional transcriptional activity or the expression of LSF (e.g., suchas protein level of LSF, and/or gene transcript level of LSF), by atleast about 10%, at least about 15%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, 95%, 99% oreven 100%, as compared to the expression in the absence of inhibitors ofLSF. The expression of LSF can be measured by standard methods known toan skilled artisan such as western blot, ELISA, and quantitative PCR aswell as the methods provided in Examples section.

In some embodiments, inhibitors of LSF as disclosed herein can inhibitor decrease cellular LSF activity by at least about 10%, relative to theactivity level in the absence of inhibitors of LSF, e.g., at least about15%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, 95%, 99% or even 100%. In certain embodiments,inhibitors of LSF as disclosed herein can decrease expression ofdownstream genes up-regulated by LSF, e.g. SPP1 encoding OPN, by aboutat least 10%, at least about 15%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, 95%, 99% oreven 100%, as compared to the expression in the absence of inhibitors ofLSF. In alternative embodiments, inhibitors of LSF can increaseexpression of downstream genes down-regulated by LSF, e.g. TAGLN, byabout at least 10%, at least about 15%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, 95%, 99% oreven 100%, as compared to the expression in the absence of inhibitors ofLSF.

As disclosed herein, the inventors have identified compounds that caninhibit LSF, such compounds being represented by formula (I), whereinformula (I) has the structure:

In formula (I), R¹, R², R³ and R⁴ are each independently selected fromthe group of hydrogen, C₁-C₆ alkyl, C₁-C₆haloalkyl, C₁-C₆ heteroalkyl,OR^(3A), SR^(3A), SO₂R^(3A), NR^(3A)R^(4A), PO_(m)(R^(3A))_(n), F, Br,Cl, I, heteroaryl, or aryl, wherein the alkyl, haloalkyl, heteroalkyl,heteroaryl, and aryl can be optionally substituted with halogen, C₁-C₄alkyl, C₁-C₄ haloalkyl, C₁-C₄ heteroalkyl, di(C₁-C₂₄alkyl)amino, orC₁-C₆ alkoxy. In various embodiments, R² and R³ together can form asecond bond between the carbons to which they are attached.

R⁵ is selected from the group of hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl,C₁-C₆ heteroalkyl, aryl, heteroaryl, F, Br, Cl, or I.

R⁶ and R⁷ are each independently selected from the group of hydrogen, F,Br, Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A), SO₂R^(3A),PO_(m)(R^(3A))_(n), C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,aryl or heteroaryl.

R⁸ and R⁹ are each independently selected from the group of hydrogen,C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ heteroalkyl, OR^(3A), SR^(3A),SO₂R^(3A), NR^(3A)R^(4A), PO_(m)(R^(3A))_(n), F, Br, Cl, I, heteroarylor aryl.

In various embodiments, R⁹ is not NR^(3A)R^(4A). In various embodiments,R⁹ is not an aryl, heterocycyl or halogen.

R^(3A) and R^(4A) are each independently selected from the group ofhydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl,C₁-C₈ heteroalkyl, heteroaryl, or aryl, wherein the alkyl, alkenyl,alkynyl, haloalkyl, heteroalkyl, heteroaryl, and aryl can be optionallysubstituted with halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄heteroalkyl or C₁-C₆ alkoxy.

m and n are each independently selected from 1, 2, or 3.

In certain embodiments of formula (I), R^(1′), R³ and R⁴ are hydrogen.R² is an optionally substituted aryl, for example

wherein R^(5A) is C₁-C₆ alkyl. R⁵ can be hydrogen or C₁-C₆ alkyl, suchas —CH₃. R⁶ is hydrogen. R⁷ can be hydrogen or —OR^(5A), wherein R^(5A)is C₁-C₆ alkyl, e.g. CH₃. R⁸ can be hydrogen or —OR^(5A), and R⁹ is—OR^(5A), wherein R^(5A) is C₁-C₆ alkyl, e.g. CH₃. In some embodiments,R⁸ and R⁹ together with the carbons to which they are attached form anoptionally substituted 5-membered heterocycyl, such as

wherein X is hydrogen or halogen such as fluorine. In variousembodiments, R⁹ is not NR^(3A)R^(4A). In various embodiments, R⁹ is notan aryl, heterocycyl or halogen.

In some embodiments, inhibitors of LSF with formula (I) can berepresented by formula (II), wherein the formula (II) has the structure:

In formula (II), R¹, R², R³ and R⁴ are each independently selected fromthe group of hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,OR^(3A), R^(3A), SR^(3A), SO₂R^(3A), NR^(3A)R^(4A), PO_(m)(R^(3A))_(n),F, Br, Cl, I, heteroaryl, or aryl, wherein the alkyl, haloalkyl,heteroalkyl, heteroaryl, and aryl can be optionally substituted withhalogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ heteroalkyl,di(C₁-C₂₄alkyl)amino, or C₁-C₆ alkoxy. In various embodiments, R² and R³together can form a second bond between the carbons to which they areattached. In some embodiments, R² is a substituted aryl. In someembodiments, R² is a substituted phenyl. In some embodiments, asubstituted phenyl is a C₁-C₆ alkoxy substituted phenyl. In certainembodiments, a C₁-C₆ alkoxy substituted phenyl, or OMe, OEt, O^(n)Pr,O^(i)Pr, O^(n)Bu, O^(i)Bu or O^(t)Bu.

R⁵ is selected from the group of hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl,C₁-C₆ heteroalkyl, aryl, heteroaryl, F, Br, Cl, or I.

R⁶ and R⁷ are each independently selected from the group of hydrogen, F,Br, Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A), SO₂R^(3A),PO_(m)(R^(3A))_(n), C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,aryl or heteroaryl. In some embodiments, R⁷ is OR^(3A). In someembodiments, OR^(3A) is O—C₁-C₈ alkyl.

R⁸ is selected from the group of hydrogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl,C₁-C₄ heteroalkyl, OR^(3A), SR^(3A), SO₂R^(3A), NR^(3A)R^(4A),PO_(m)(R^(3A))_(n), F, Br, Cl, I, heteroaryl or aryl. In someembodiments, OR^(3A) is O—C₁-C₈ alkyl. In some embodiments, C₁-C₈ alkylis a branched chain alkyl. In some embodiments, C₁-C₈ alkyl is methyl,ethyl, n-propyl, i-propyl, n-butyl, i-butyl, or t-butyl.

R^(3A) and R^(4A) are each independently selected from the group ofhydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl,C₁-C₈ heteroalkyl, heteroaryl, or aryl, wherein the alkyl, alkenyl,alkynyl, haloalkyl, heteroalkyl, heteroaryl, and aryl can be optionallysubstituted with halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄heteroalkyl or C₁-C₆ alkoxy.

In some embodiments, R^(3A) is C₁-C₈ alkyl. In some embodiments, R^(3A)is C₁-C₈ alkyl is a straight chain alkyl. In some embodiments, C₁-C₈alkyl is a branched chain alkyl. In some embodiments, C₁-C₈ alkyl ismethyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, or t-butyl.

In some embodiments, OR^(3A) is O—C₁-C₄ alkyl. In some embodiments,C₁-C₈ alkyl is a straight chain alkyl. In some embodiments, C₁-C₈ alkylis a branched chain alkyl. In some embodiments, C₁-C₈ alkyl is methyl,ethyl, n-propyl, i-propyl, n-butyl, i-butyl, or t-butyl.

m is 1, 2, or 3; and n is 1, 2, or 3;

In some embodiments of formula (II), R^(1′), R³ and R⁴ are hydrogen. R²is an optionally

substituted aryl, for example, wherein R^(5A) is C₁-C₆ alkyl. R⁵ can behydrogen or C₁-C₆ alkyl, such as —CH₃. R⁶ is hydrogen. R⁷ can behydrogen or —OR^(5A), wherein R^(5A) is C₁-C₆ alkyl, e.g. CH₃. R⁸ can behydrogen or —OR^(5A), wherein R^(5A) is C₁-C₆ alkyl, e.g. CH₃, andR^(3A) is C₁-C₆ alkyl, e.g. CH₃. In some embodiments, R⁸ and OR^(3A)together with the carbons to which they are attached form an optionallysubstituted 5-membered heterocycyl, such as

wherein X is hydrogen or halogen such as fluorine.

In some embodiments, inhibitors of LSF with formula (I) can berepresented by formula (III), wherein the formula (III) has thestructure:

In formula (III), R¹, R², R³ and R⁴ are each independently selected fromthe group of hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,OR^(3A)R^(3A), SR^(3A), SO₂R^(3A), NR^(3A)R^(4A), PO_(m)(R^(3A))_(n), F,Br, Cl, I, heteroaryl, or aryl, wherein the alkyl, haloalkyl,heteroalkyl, heteroaryl, and aryl can be optionally substituted withhalogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ heteroalkyl,di(C₁-C₂₄alkyl)amino, or C₁-C₆ alkoxy. In various embodiments, R² and R³together can form a second bond between the carbons to which they areattached.

R⁵ is selected from the group of hydrogen, C₁-C₆ alkyl, C₁-C₆haloalkyl,C₁-C₆ heteroalkyl, aryl, heteroaryl, F, Br, Cl, or I;

R⁶ and R⁷ are each independently selected from the group of hydrogen, F,Br, Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A), SO₂R^(3A),PO_(m)(R^(3A))_(n), C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,aryl or heteroaryl; R¹⁰ and R¹¹ are each independently selected from thegroup of hydrogen, F, Br, Cl, or I.

R^(3A) and R^(4A) are each independently selected from the group ofhydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl,C₁-C₈ heteroalkyl, heteroaryl, or aryl, wherein the alkyl, alkenyl,alkynyl, haloalkyl, heteroalkyl, heteroaryl, and aryl can be optionallysubstituted with halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄heteroalkyl or C₁-C₆ alkoxy.

m is 1, 2, or 3; and n is 1, 2, or 3;

In some embodiments of formula (III), R^(1′), R³ and R⁴ are hydrogen. R²is an optionally substituted aryl, for example,

wherein R^(5A) is C₁-C₆ alkyl. R⁵ can be hydrogen or C₁-C₆ alkyl, suchas —CH₃. R⁶ is hydrogen. R⁷ can be hydrogen or —OR^(5A), wherein R^(5A)is C₁-C₆ alkyl, e.g. CH₃. R¹⁰ and R¹¹ are each independently selectedfrom either hydrogen or halogen such as fluorine.

In some embodiments, inhibitors of LSF with formula (III) can berepresented by formula (XXVI), wherein the formula (XXVI) has thestructure:

In formula (XXVI), R², R³ and R⁴ are each independently selected fromthe group of hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl,OR^(3A), SR^(3A), SO₂R^(3A), NR^(3A)R^(4A), PO_(m)(R^(3A))_(n), F, Br,Cl, I, heteroaryl, or aryl, wherein the alkyl, haloalkyl, heteroalkyl,heteroaryl, and aryl can be optionally substituted with halogen, C₁-C₄alkyl, C₁-C₄ haloalkyl, C₁-C₄ heteroalkyl or C₁-C₆ alkoxy. In variousembodiments, R² and R³ together can form a second bond between thecarbons to which they are attached.

R⁵ is selected from the group of hydrogen, C₁-C₆ alkyl, C₁-C₆haloalkyl,C₁-C₆ heteroalkyl, aryl, heteroaryl, F, Br, Cl, or I;

R⁶, R⁷, R¹³, R¹⁴, R¹⁵ and R^(15′) are each independently selected fromthe group of hydrogen, F, Br, Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A),SO₂R^(3A), PO_(m)(R^(3A))_(n), C₁-C₆ alkyl, C₂-C₈ alkenyl, C₁-C₆haloalkyl, C₁-C₆ heteroalkyl, aryl or heteroaryl;

each R¹² is independently selected from the group of hydrogen, F, Br,Cl, I, OR^(3A), NR^(3A)R^(4A), SR^(3A), SO₂R^(3A), PO_(m)(R^(3A))_(n),C₁-C₆ alkyl, C₂-C₈ alkenyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl, aryl orheteroaryl;

R¹⁶ and R¹⁷ are each independently selected from the group of hydrogen,F, Br, Cl, I, C₁-C₄ alkyl, or OR^(3A);

R^(3A) and R^(4A) are each independently selected from the group ofhydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl,C₁-C₈ heteroalkyl, heteroaryl, or aryl, wherein the alkyl, alkenyl,alkynyl, haloalkyl, heteroalkyl, heteroaryl, and aryl can be optionallysubstituted with halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄heteroalkyl or C₁-C₆ alkoxy.

m is 1, 2, or 3; and n is 1, 2, or 3;

In certain embodiments of formula (XXVI), R², R³ and R⁴ can be hydrogen,—CH₃, —CH₂CH₃ or R⁴ is hydrogen or —CH₃ and R² and R³ together can forma second bond between the carbons to which they are attached. R⁵ can behydrogen, —CH₃ or —CH₂CH₃. R⁶, R¹³, R¹⁴ and R^(15′) each independentlycan be hydrogen, F, Cl, Br, I, —CH₃ or —CH₂CH₃. R⁷ can be hydrogen, F,Cl, Br, I, —CH₃, —CH₂CH₃ or —OR^(5A), wherein R^(5A) is —CH₃ or —CH₂CH₃.R¹² can be hydrogen, F, Br, Cl, —CH═CH—(CH₂)₀₋₅CH₃, NR^(3A)R^(4A) or—OR^(5A), wherein R^(3A), R^(4A) and R^(5A) are independently a C₁-C₆alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl orsec-butyl. R¹⁵ can be OR^(3A), where R^(3A) is a C₁-C₆ alkyl such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl or sec-butyl. R¹⁶and R¹⁷ are each independently selected from either hydrogen or halogensuch as fluorine.

In some embodiments, R^(3A) is OR^(3A). In some embodiments, OR^(3A) isC₁-C₈ alkyl. In some embodiments, C₁-C₈ alkyl is a straight chain alkyl.In some embodiments, C₁-C₈ alkyl is a branched chain alkyl. In someembodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl,i-butyl, or t-butyl.

In some embodiments, R¹⁶ and R¹⁷ are different. In some embodiments, R¹⁶and R¹⁷ are the same. In some embodiments, at least one of R¹⁶ or R¹⁷ isF. In some embodiments both R¹⁶ and R¹⁷ is F.

In some embodiments, R¹⁵ is OR^(3A). In some embodiments, R^(3A) isC₁-C₈ alkyl. In some embodiments, C₁-C₈ alkyl is a straight chain alkyl.In some embodiments, C₁-C₈ alkyl is a branched chain alkyl. In someembodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl,i-butyl, or t-butyl.

In some embodiments, the R¹ group in Formulas (I), (II), (III) or (XXVI)is a phenyl substituted with at least one C₁-C₆ alkoxyl and at least onedi(C₁-C₂₄alkyl)amino. In some embodiments, R¹ is

wherein R²¹ is C₁-C₆alkyl and R²² and R²³ are independently selectedC₁-C₂₄alkyl. In some embodiments, R¹ is

In some preferred embodiments, R¹ is

In some embodiments, the R¹ group in Formulas (I), (II), (III) or (XXVI)is a phenyl substituted with at least one C₁-C₆ alkoxyl and at least onehalogen. In some embodiments, R^(1′) is

wherein R²¹ is C₁-C₆alkyl and R²⁴ is halogen. In some embodiments, is

In some preferred embodiments, R^(1′) is

In some embodiments, the R¹ group in Formulas (I), (II), (III) or (XXVI)is a phenyl substituted with at least one C₁-C₆ alkoxyl and at least oneC₂-C₆alkenyl. In some embodiments, R^(1′) is

wherein R²¹ is C₁-C₆alkyl and R²⁵ is C₂-C₈alkenyl. In some embodiments,R^(1′) is

In some preferred embodiments, R^(1′) is

In one embodiment, an inhibitor of LSF of formula (I), (II), (III) or(XXVI) is a compound of formula (IV), wherein the formula (IV) has thestructure:

An inhibitor of LSF with the formula (IV) is known in the art under achemical name,8-(2-ethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one. Itis also known in the art under an alternative name,8-(2ethoxyphenyl)-2H,5H,6H,7H,8H-[1,3]dioxolo[4,5-g]quinolin-6-one. Thesimplified molecular input line entry specification (SMILES) of thecompound of formula (IV) is known in the art asCCOC(C═CC═C1)=C1C(C2=CC(OCO3)=C3C═C2N4)CC4=O. The IUPAC InternationalChemical Identifier (InCHI) of the compound of formula (IV) known to anskilled artisan is1S/C18H17NO4/c1-2-21-15-6-4-3-5-11(15)12-8-18(20)19-14-9-17-16(7-13(12)14)22-10-23-17/h3-7,9,12H,2,8,10H2,1H3,(H,19,20).

In some embodiments, inhibitors of LSF of formula (I), (II), (III) or(XXVI) can be a compound selected from the group consisting of compoundsof formula (V) to (XXV):

As disclosed herein, the inventors have identified compounds that caninhibit LSF. In some embodiments, such compounds being represented byformula (III′):

or enantiomers, prodrugs, derivatives, and pharmaceutically acceptablesalts thereof.

In compounds of Formula (III′), R^(1′) is an aryl substituted with atleast one C₁-C₆alkoxyl and at least one di(C₁-C₂₄alkyl)amino, halogen,C₁-C₆alkyl, C₂-C₆alkenyl, polyethylene glycol or polyethylene glycolsubstituted C₁-C₆alkyl, wherein the substituted aryl can be optionallyfurther substituted with halogen, C₁-C₄alkyl, C₁-C₄haloalkyl, C₁-C₄heteroalkyl, di(C₁-C₂₄alkyl)amino or combinations thereof.

In some compounds of Formula (III′), R^(1′) is an aryl substituted withat least one C₁-C₆ alkoxyl and at least one di(C₁-C₂₄alkyl)amino,halogen, or C₂-C₆alkenyl, polyethylene glycol or polyethylene glycolsubstituted C₁-C₆alkyl, wherein the substituted aryl can be optionallyfurther substituted with halogen, C₁-C₄alkyl, C₁-C₄haloalkyl, C₁-C₄heteroalkyl, di(C₁-C₂₄alkyl)amino or combinations thereof.

In some compounds of Formula (III′), R^(1′) is an aryl substituted withat least one C₁-C₆ alkoxyl and at least one di(C₁-C₂₄alkyl)amino,halogen or C₂-C₆alkenyl, wherein the substituted aryl can be optionallyfurther substituted with halogen, C₁-C₄alkyl, C₁-C₄haloalkyl, C₁-C₄heteroalkyl, di(C₁-C₂₄alkyl)amino or combinations thereof.

In some compounds of Formula (III′), R^(1′) is an aryl substituted withat least one C₁-C₆ alkoxyl and at least one di(C₁-C₂₄alkyl)amino,wherein the substituted aryl can be optionally further substituted withhalogen, C₁-C₄alkyl, C₁-C₄haloalkyl, C₁-C₄ heteroalkyl,di(C₁-C₂₄alkyl)amino or combinations thereof.

In some compounds of Formula (III′), R^(1′) is an aryl substituted withat least one C₁-C₆ alkoxyl and at least one halogen, wherein thesubstituted aryl can be optionally further substituted with halogen,C₁-C₄alkyl, C₁-C₄haloalkyl, C₁-C₄ heteroalkyl, di(C₁-C₂₄alkyl)amino orcombinations thereof.

In some compounds of Formula (III′), R^(1′) is an aryl substituted withat least one C₁-C₆ alkoxyl and at least one C₂-C₆alkenyl, wherein thesubstituted aryl can be optionally further substituted with halogen,C₁-C₄alkyl, C₁-C₄haloalkyl, C₁-C₄ heteroalkyl, di(C₁-C₂₄alkyl)amino orcombinations thereof.

In some compounds of Formula (III′), R^(1′) is an aryl substituted withat least one C₁-C₆ alkoxyl and at least one of halogen, C₁-C₆alkyl,C₂-C₈alkenyl, C₁-C₄haloalkyl, C₁-C₄ heteroalkyl, di(C₁-C₂₄alkyl)amino,polyethylene glycol, or combinations thereof.

In some embodiments, the aryl of the R¹ group is substituted with theC₁-C₆alkoxyl at the ortho position.

In compounds of Formula (III′), where the aryl of R¹ group issubstituted with a C₁-C₆alkoxyl and a second substituent. Withoutlimitations the second substituent can be present at the meta, ortho orpara position. In some embodiments, the C₁-C₆alkoxyl is at the orthoposition and the second substituent is at the para position.

In some embodiments, R^(1′) is a phenyl substituted with at least oneC₁-C₆ alkoxyl and at least one di(C₁-C₂₄alkyl)amino. In someembodiments, R^(1′) is

wherein R²¹ is C₁-C₆alkyl and R²² and R²³ are independently selectedC₁-C₂₄alkyl. Exemplary alkyls for the R²¹ group include, but are notlimited to, methyl, ethyl, propyl, isopropyl, 1-butyl, 2-butyl,2-methylpropyl, pentyl, t-butyl, and hexyl. In some embodiments, R²¹ isethyl.

Without limitations, R²² and R²³ can be same or different. Moreover,they can comprise the same number of carbons or different number ofcarbons. In some embodiments, R²² and R²³ are selected independentlyfrom C₁-C₆ alkyl groups. Exemplary alkyls for the R²² and R²³ groupinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,1-butyl, 2-butyl, 2-methylpropyl, pentyl, t-butyl, and hexyl. In someembodiments, R²² and R²³ are methyl.

In some embodiments, R^(1′) is

In some embodiments, R^(1′) is

In some embodiments, R^(1′) is a phenyl substituted with at least oneC₁-C₆ alkoxyl and at least one halogen. In some embodiments, R^(1′) is

wherein R²¹ is C₁-C₆alkyl and R²⁴ is halogen. Exemplary alkyls for theR²¹ group include, but are not limited to, methyl, ethyl, propyl,isopropyl, 1-butyl, 2-butyl, 2-methylpropyl, pentyl, t-butyl, and hexyl.In some embodiments, R²¹ is ethyl. In some embodiments, R²⁴ is selectedfrom the group consisting of Br, F, and Cl.

In some embodiments, R^(1′) is

In some embodiments, R^(1′) is

In some embodiments, R^(1′) is a phenyl substituted with at least oneC₁-C₆ alkoxyl and at least one C₂-C₆alkenyl. In some embodiments, R^(1′)is

wherein R²¹ is C₁-C₆alkyl and R²⁵ is C₂-C₆alkenyl. Exemplary alkyls forthe R²¹ group include, but are not limited to, methyl, ethyl, propyl,isopropyl, 1-butyl, 2-butyl, 2-methylpropyl, pentyl, t-butyl, and hexyl.In some embodiments, R²¹ is ethyl. In some embodiments, R²⁵ is selectedfrom the group consisting of ethylenyl, propylenyl, butylenyl,isobutylenyl, and pentylenyl. In some embodiments, R²⁵ is pentylenyl.

In some embodiments, R^(1′) is

In some embodiments, R^(1′) is

In compounds of Formula (III′), R² and R³ are hydrogen or R² and R³together form a second bond between the carbons to which they areattached. In some embodiments, R² and R³ are hydrogen.

In compounds of Formula (III′) R⁴ is hydrogen and R⁵ is selected fromthe group consisting of hydrogen and C₁-C₆ alkyl. Exemplary alkyls forthe R⁵ group include, but are not limited to, methyl, ethyl, propyl,isopropyl, 1-butyl, 2-butyl, 2-methylpropyl, pentyl, t-butyl, and hexyl.In some embodiments, R⁵ is hydrogen or methyl. In some furtherembodiments, R⁵ is hydrogen.

In compounds of Formula (III′), R⁶ and R⁷ are each independentlyselected from the group consisting of hydrogen, F, Br, Cl, and I.Without limitations, R⁶ and R⁷ can be the same or different. In someembodiments, R⁶ and R⁷ are hydrogen.

In compounds of Formula (III′), R¹⁰ and R¹¹ are each independentlyselected from the group consisting of hydrogen, F, Br, Cl, and I.Without limitations, R¹⁰ and R¹¹ can be the same or different. In someembodiments, R⁶ and R⁷ are hydrogen of fluorine. In some embodiments R¹⁰and R¹¹ are hydrogen. In some other embodiments, R¹⁰ and R¹¹ arefluorine.

An exemplary compound of Formula (III′) is the compound FQI-34represented having the structure:

In some embodiments, a compound of Formula (III′) can be selected fromthe group consisting of

In some embodiments, a LSF inhibitor is not FQI-F, FQI32, FQI33 orPEG-FQI.

A compound of Formula (III′) can be formulated in a pharmaceuticalcomposition described herein. Further, a compound of Formula (III′) canbe used in the methods, e.g., a method for inhibiting LSF or treatingcancer disclosed herein.

All structures of any of formula (I) to (XXVI) and (III′) are providedherein for illustrative purpose and disclose a particular isomer.However, one of ordinary skill in the art will recognize all possibleisomers of the structures of any of formula (I) to (XXVI) and (III′).Therefore, other isomers such as enantiomers of any of formula (I) to(XXVI) and (III′) are considered to fall within the scope of theinvention. As used herein, the term “isomer” refers to a compound havingthe same molecular formula but differing in structure. Isomers whichdiffer only in configuration and/or conformation are referred to as“stereoisomers.” The term “isomer” is also used to refer to anenantiomer.

The term “enantiomer” is used to describe one of a pair of molecularisomers which are mirror images of each other and non-superimposable.For example, as disclosed herein, compounds C1-1 and C1-2 as shown inFIG. 1 are enantiomers of compounds of formula (IV). The designations“R” and “S” of compounds C1-1 and C1-2 in FIG. 1, respectively, are usedto denote the absolute configuration of the molecule about its chiralcenter. The designations may appear as a prefix or as a suffix; they mayor may not be separated from the isomer by a hyphen; they may or may notbe hyphenated; and they may or may not be surrounded by parentheses. Thedesignations “(+)” and “(−)” of compounds C1-1 and C1-2 in FIG. 1 areemployed to designate the sign of rotation of plane-polarized light bythe compound, with (−) meaning that the compound is levorotatory(rotates to the left). A compound prefixed with (+) is dextrorotatory(rotates to the right). Other terms used to designate or refer toenantiomers include “stereoisomers” (because of the differentarrangement or stereochemistry around the chiral center; although allenantiomers are stereoisomers, not all stereoisomers are enantiomers) or“optical isomers” (because of the optical activity of pure enantiomers,which is the ability of different pure enantiomers to rotateplanepolarized light in different directions). Enantiomers generallyhave identical physical properties, such as melting points and boilingpoints, and also have identical spectroscopic properties. Enantiomerscan differ from each other with respect to their interaction withplane-polarized light and with respect to biological activity.

In various embodiments, inhibitors of LSF of any of formula (I) to(XXVI) and (III′) include enantiomers, derivatives, prodrugs, andpharmaceutically acceptable salts thereof.

The term “derivative” as used herein refers to a chemical substancerelated structurally to another, i.e., an “original” substance, whichcan be referred to as a “parent” compound. A “derivative” can be madefrom the structurally-related parent compound in one or more steps. Thegeneral physical and chemical properties of a derivative are alsosimilar to the parent compound.

In some embodiments, prodrugs of LSF inhibitors selected from any offormula (I) to (XXVI) also fall within the scope of the invention. Asused herein, a “prodrug” refers to a compound that can be converted viasome chemical or physiological process (e.g., enzymatic processes andmetabolic hydrolysis) to a LSF inhibitor selected from the groupconsisting of compounds of formula (I) to (XXVI) and (III′).

Thus, the term “prodrug” also refers to a precursor of a biologicallyactive compound that is pharmaceutically acceptable. A prodrug may beinactive when administered to a subject, i.e. an ester, but is convertedin vivo to an active compound, for example, by hydrolysis to the freecarboxylic acid or free hydroxyl. The prodrug compound often offersadvantages of solubility, tissue compatibility or delayed release in anorganism. The term “prodrug” is also meant to include any covalentlybonded carriers, which release the active compound in vivo when suchprodrug is administered to a subject. Prodrugs of an active compound maybe prepared by modifying functional groups present in the activecompound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent active compound. Prodrugsinclude compounds wherein a hydroxy, amino or mercapto group is bondedto any group that, when the prodrug of the active compound isadministered to a subject, cleaves to form a free hydroxy, free amino orfree mercapto group, respectively. Examples of prodrugs include, but arenot limited to, acetate, formate and benzoate derivatives of an alcoholor acetamide, formamide and benzamide derivatives of an amine functionalgroup in the active compound and the like. See Harper, “DrugLatentiation” in Jucker, ed. Progress in Drug Research 4:221-294 (1962);Morozowich et al, “Application of Physical Organic Principles to ProdrugDesign” in E. B. Roche ed. Design of Biopharmaceutical Propertiesthrough Prodrugs and Analogs, APHA Acad. Pharm. Sci. 40 (1977);Bioreversible Carriers in Drug in Drug Design, Theory and Application,E. B. Roche, ed., APHA Acad. Pharm. Sci. (1987); Design of Prodrugs, H.Bundgaard, Elsevier (1985); Wang et al. “Prodrug approaches to theimproved delivery of peptide drug” in Curr. Pharm. Design. 5(4):265-287(1999); Pauletti et al. (1997) Improvement in peptide bioavailability:Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev.27:235-256; Mizen et al. (1998) “The Use of Esters as Prodrugs for OralDelivery of (3-Lactam antibiotics,” Pharm. Biotech. ll:345-365;Gaignault et al. (1996) “Designing Prodrugs and Bioprecursors I. CarrierProdrugs,” Pract. Med. Chem. 671-696; Asgharnejad, “Improving Oral DrugTransport”, in Transport Processes in Pharmaceutical Systems, G. L.Amidon, P. I. Lee and E. M. Topp, Eds., Marcell Dekker, p. 185-218(2000); Balant et al., “Prodrugs for the improvement of drug absorptionvia different routes of administration”, Eur. J. Drug Metab.Pharmacokinet., 15(2): 143-53 (1990); Balimane and Sinko, “Involvementof multiple transporters in the oral absorption of nucleosideanalogues”, Adv. 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Do theyhave advantages in clinical practice?”, Drugs 29(5): 455-73 (1985); Tanet al. “Development and optimization of anti-HIV nucleoside analogs andprodrugs: A review of their cellular pharmacology, structure-activityrelationships and pharmacokinetics”, Adv. Drug Delivery Rev. 39(1-3):117-151 (1999); Taylor, “Improved passive oral drug delivery viaprodrugs”, Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino andBorchardt, “Prodrug strategies to enhance the intestinal absorption ofpeptides”, Drug Discovery Today 2(4): 148-155 (1997); Wiebe and Knaus,“Concepts for the design of anti-HIV nucleoside prodrugs for treatingcephalic HIV infection”, Adv. Drug Delivery Rev.: 39(1-3):63-80 (1999);Waller et al., “Prodrugs”, Br. J. Clin. Pharmac. 28: 497-507 (1989),content of all of which is herein incorporated by reference in itsentirety.

Inhibitors of LSF of formula (I) to (XXVI) and (III′) also includepharmaceutically acceptable salts thereof. As used herein, the term“pharmaceutically-acceptable salts” refers to the conventional nontoxicsalts or quaternary ammonium salts of LSF inhibitors as disclosedherein, e.g., from non-toxic organic or inorganic acids. These salts canbe prepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting a LSF inhibitor in itsfree base or acid form with a suitable organic or inorganic acid orbase, and isolating the salt thus formed during subsequent purification.Conventional nontoxic salts include those derived from inorganic acidssuch as sulfuric, sulfamic, phosphoric, nitric, and the like; and thesalts prepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isothionic, and the like. See, for example,Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19 (1977),content of which is herein incorporated by reference in its entirety.

In some embodiments of the aspects described herein, representativepharmaceutically acceptable salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,succinate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like.

Inhibition of LSF has been previously discussed as a potential treatmentof latent HIV infection or cancer in general, or as a therapeuticregulator of immune function, specifically when there is a need thereofto decrease inflammatory response (U.S. Patent Application No.: US2009/0081183 and International Patent Application No.: WO1998/36641,which are incorporated herein in their entirety by reference). However,these patent applications do not teach or describe any small-moleculeLSF inhibitors of the invention as disclosed herein, or the use thereoffor treatment of hepatocellular carcinoma (HCC).

Use of the LSF Inhibitors of any of Formula (I) to (XXVI) and (III) toTreat Cancers and Hepatocellular Carcinoma

In some embodiments, a LSF inhibitor compound of any of formulas (I) to(XXVI) and (III′) as disclosed herein can be used to treat variouscancers, such as liver cancer (hepatocellular carcinoma), brain cancer,breast cancer, colon cancer, head and neck squamous cell carcinoma, lungcancer, pancreatic cancer, ovarian cancer, and thyroid cancer; HIV;inflammation-related diseases such as hepatitis B virus (HBV), hepatitisC (HCV), cirrhosis and Alzheimer's disease. In some embodiments, theliver diseases can be any selected from, but not limited to, HBV, HCV,cirrhosis, hepatic adenoma, hepatic angiosarcoma and hepaticangiosarcomas; emphysema; and hereditary hemochromatosis.

In some embodiments, a LSF inhibitor compound of any of formulas (I) to(XXVI) and (III′) as disclosed herein can be used to treat othercancers, for example, cervical cancer, colon cancers, melanomas and thelike. Other cancers which can be treated include any cancer withoverexpression of LSF in the tumor, for example, but not limited to,oligodendroglioma, meningioma, GBM, breast cancer, colon cancer,Non-Hodgkin's small cell carcinoma (HNSCC), lung cancer(adrenocarcinomas), lung cancer (small cell carcinoma), pancreaticcancer, ovarian cancer, thyroid cancer and undifferentiated cancer.

In some embodiments, a LSF inhibitor of any of formulas (I) to (XXVI)and (III′) as disclosed herein can be used to treat any cancer celltype. Cancers include, but are not limited to, bladder cancer; breastcancer; brain cancer including glioblastomas and medulloblastomas;cervical cancer; choriocarcinoma; colon cancer including colorectalcarcinomas; endometrial cancer; esophageal cancer; gastric cancer; headand neck cancer; hematological neoplasms including acute lymphocytic andmyelogenous leukemia, multiple myeloma, AIDS associated leukemias andadult T-cell leukemia lymphoma; intraepithelial neoplasms includingBowen's disease and Paget's disease, liver cancer; lung cancer includingsmall cell lung cancer and non-small cell lung cancer; lymphomasincluding Hodgkin's disease and lymphocytic lymphomas; neuroblastomas;oral cancer including squamous cell carcinoma; osteosarcomas; ovariancancer including those arising from epithelial cells, stromal cells,germ cells and mesenchymal cells; pancreatic cancer; prostate cancer;rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma,liposarcoma, fibrosarcoma, synovial sarcoma and osteosarcoma; skincancer including melanomas, Kaposi's sarcoma, basocellular cancer, andsquamous cell cancer; testicular cancer including germinal tumors suchas seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors,and germ cell tumors; thyroid cancer including thyroid adenocarcinomaand medullar carcinoma; transitional cancer and renal cancer includingadenocarcinoma and Wilm's tumor.

In one embodiment, compositions and methods of the invention asdisclosed herein is used to treat a subject with hepatocellularcarcinoma (HCC).

In another embodiment, a subject at high risk of developing HCC issuitable for treatment with the compositions of the invention comprisingat least a LSF inhibitor as disclosed herein.

Hepatocellular carcinoma (HCC) is one of the five most common cancersworldwide (1). The incidence of HCC is increasing despite a decrease inoverall incidence of all cancers (2, 3). In the United States, theestimated new cases of HCC for 2008 were 21,370, of which 18,410 wereexpected to die (2). The mean 5-year survival rate is less than 10%. Themortality rate of HCC parallels that of its incidence because HCC is atumor with rapid growth and early vascular invasion that is resistant toconventional chemotherapy, and no systemic therapy is available for theadvanced disease (4).

To date, other than curative resection, treatments for HCC have hadminimal impact on survival. Unfortunately, approximately 90% of HCCpatients have unresectable HCC. Moreover, even after potentiallycurative hepatectomy in patients with resectable HCC, new HCC arises inthe cirrhotic remnants in 70% of these patients, and frequently arisesin the grafted liver following orthotopic liver transplantation. Otherapproaches to treating HCC, such as intralesional ethanol injection,chemoembolization, radiofrequency ablation, cryosurgery and radiationtherapy have demonstrated some success in selected patient populations;however, the efficacies of these approaches have not been definitelyestablished. Both percutaneous intralesional ethanol injection andtransarterial chemoembolization have shown limited success, but notwithout risks of serious side effects. Radiotherapy is not usually anoption because liver is very radiosensitive. Systemic chemotherapy forHCC has had uniformly poor response rates, and therefore it is generallyreserved for patients ineligible for curative resection.

All systemic therapies for HCC to date are associated with uniformlypoor outcomes, and only one chemotherapeutic agent (sorafenib), alone orin combination with other treatments, has been associated with anyimprovement in survival rates (Fuchs et al., 94 Cancer 3186 (2002)). Inaddition, most patients with HCC have underlying liver disease so theirability to tolerate to undergo surgery is compromised. Therefore, thereis a strong need in the art to provide improved methods for treatment ofHCC.

One aspect of the invention provides methods for therapeutic andprophylactic treatment of cancers, e.g., HCC by administering to asubject a pharmaceutical composition comprising an effective amount of aLSF inhibitor. In preferred embodiments, a LSF inhibitor is a smallmolecule selected from the group consisting of compounds of formulae (I)to (XXVI) and (III′), and enantiomers, prodrugs, derivatives, andpharmaceutically acceptable salts thereof. In one embodiment, a LSFinhibitor is a compound of formula (IV) or enantiomers, prodrugs,derivatives or pharmaceutically acceptable salts thereof. In otherembodiments, a LSF inhibitor is a compound with any of formula (V) to(XXVI) or enantiomers, prodrugs, derivatives or pharmaceuticallyacceptable salts thereof. In some embodiments, a LSF inhibitor is thecompound FQI-34 or enantiomers, prodrugs, derivatives orpharmaceutically acceptable salts thereof. In some embodiments, a LSFinhibitor is the compound FQI-Br, FQI-F, FQI-Cl, FQI32 or enantiomers,prodrugs, derivatives or pharmaceutically acceptable salts thereof.

The inventors in collaboration with other scientists have revealed thatadministration of of a LSF inhibitor formula (IV) or (III″) at about1.0-2.0 mg/kg once every 3 days, can suppress the growth of HCC tumorsize by 65-80% in an in vivo mouse model, as compared to the micewithout treatment of an LSF inhibitor (data not shown). Accordingly, insome embodiments, individuals with heptocellular carcinoma can betreated with a LSF inhibitor of any of compounds of formula (I) to(XXVI) and (III′) as disclosed herein and a pharmaceutical compositionthereof.

In some embodiments, a LSF inhibitor or a pharmaceutical compositionthereof as disclosed herein can be used in conjunction with othertherapeutic treatment of HCC such as hepatointralesional ethanolinjection, chemoembolization, radiofrequency ablation, cryosurgery,radiation therapy, percutaneous intralesional ethanol injection,transarterial chemoembolization, and radiotherapy.

In some embodiments, a LSF inhibitor or a pharmaceutical compositionthereof can be used to treat HCC subjects who are not responsive to anyprior treatment of HCC, or show little/no improvement from any priortreatment of HCC, e.g. continued progression or worsening of HCC. Insuch embodiments, the HCC subjects can be treated again with theprevious therapeutic method in combination with an inhibitor of LSF. Inalternative embodiments, they can be administered with a LSF inhibitoror a pharmaceutical composition thereof alone, or concurrently withalternative therapeutic methods.

It has been previously reported that LSF is a downstream gene ofastrocyte elevated gene-1 (AEG-1), which is over-expressed in >90% ofhuman HCC patients and induces chemoresistance of HCC to anchemotherapeutic agent, such as 5-fluorouracil (5-FU) (5, 6).Accordingly, in some embodiments, an inhibitor of LSF described hereincan be administered prior to, or concurrently with at least onechemotherapeutic agent such as 5-FU. Other exemplary chemotherapeuticagents include Doxorubicin, 5-FU, Paclitaxel, Irinotecan, Patupilone,Everolimus, multikinase inhibitors (Sorafenib and Sunitinib), and EGFRinhibitors (Cetuximab, Erlotinib, Gefitinib, Brivanib, Lapatinib). Inone embodiment, a LSF inhibitor or a pharmaceutical composition thereofas disclosed herein can be combined with Sorafenib for treatment of HCC.

As a prophylactic measure against HCC recurrence or metastasis, aninhibitor of LSF or a pharmaceutical composition thereof can beadministered after surgery or after aforementioned treatments for HCCwhere solid tumors have been removed or eliminated. In some embodiments,subjects with resectable HCC can be treated with an inhibitor of LSF ora pharmaceutical composition thereof after hepatectomy or livertransplantation to prevent the recurrence of HCC.

Most cases of HCC are developed from either chronic infection withhepatitis B or hepatitis C virus (HBV or HCV, respectively), or hepaticcirrhosis due to alcoholism. Chronic hepatitis can progress intocirrhosis (a noncancerous liver disease associated with fibrosis andabnormal nodules), which increases the risk of developing HCC. Subjectswith chronic hepatitis and/or cirrhosis, therefore form a high riskpopulation. Accordingly, in some embodiments, a LSF inhibitor can beused in conjunction with other therapeutic treatment for liver diseasessuch as infection with HBV, HCV or cirrhosis, as a preventive measureagainst the onset of HCC.

In some embodiments, a LSF inhibitor of any of compounds of formula (I)to (XXVI) and (III′) as disclosed herein can be administered to asubject with a high risk of developing hepatocellular carcinoma. Forexample, subjects amenable to treatment by methods and compositions asdisclosed herein, e.g. using an inhibitor of LSF, are subjects having arisk factor for HCC. Examples of risk factors for HCC include, but notlimited to, HBV, HCV, chronic alcohol consumption, exposure to aflatoxinB1 in food (which is a liver carcinogenic chemical produced by a moldcalled Aspergillus flavus after exposure of food to a hot and humidenvironment), hepatic adenoma resulted from the use of female hormones(estrogens) and protein-building (anabolic) steroids, exposure tothorotrast (a previously used contrast agent for imaging, which caused acancer of the blood vessels in the liver called hepatic angiosarcoma),hepatic angiosarcomas (resulted from a prolonged exposure to vinylchloride, a compound used in the plastics industry), hereditaryhemochromatosis (a disorder that causes the body to accumulate excessiveamounts of iron), emphysema and cirrhosis (resulted from alpha 1anti-trypsin deficiency) and hereditary tyrosinemia. In someembodiments, a LSF inhibitor can be used alone or combined with othertherapeutic treatment of the aforementioned diseases or disorders. Infurther embodiments, subjects who have been previously subjected to highrisk of developing HCC can be continually treated with an inhibitor ofLSF or a pharmaceutical composition thereof, even after they havediscontinued treatment of liver diseases such as HBV, HCV or cirrhosis.

Other indications that can be contemplated for the use of LSF inhibitorsof the invention as disclosed herein include diseases or disorders, inwhich expression and/or biological activity of LSF is up-regulated, e.g.by inflammatory cytokines (10, 11), or in which it is desirable todecrease or inhibit LSF. Non-limiting examples of such diseases ordisorders include HIV and inflammation-associated diseases includingAlzheimer's disease.

It has been previously reported that LSF activates cellsurvival-regulating pathways, such as MEK/ERK and NF-κB pathways, and isup-regulated in various cancers (see Yoo et al., PNAS, 2010, 107;8357-8362). Accordingly, in some embodiments, a LSF inhibitor disclosedherein can be used alone or in combination with chemotherapeutic agentsfor treatment of other various cancers such as brain cancer, breastcancer, colon cancer, head and neck squamous cell carcinoma, lungcancer, pancreatic cancer, ovarian cancer, and thyroid cancer. Exemplarychemotherapeutic agents include Doxorubicin, 5-FU, Paclitaxel,Irinotecan, Patupilone, Everolimus, multikinase inhibitors (Sorafeniband Sunitinib), and EGFR inhibitors (Cetuximab, Erlotinib, Gefitinib,Brivanib, Lapatinib), In some embodiments, diseases or disordersassociated with LSF-induced MEK/ERK activation can be contemplated fortreatment with a LSF inhibitor as disclosed herein alone or incombination with inhibitors of MEK/ERK pathway such as PD98059 andU0126.

Selection of Subjects for Administration with a PharmaceuticalComposition Comprising a LSF Inhibitor of any of Compounds (I) to (XXVI)and (III′)

In some embodiments, a subject amenable or suitable for treatment with acomposition comprising a LSF inhibitor of any of compounds of formula(I) to (XXVI) and (III′) as disclosed herein can be selected based on anincreased level of LSF expression in a biological sample, or tumor orcancer sample as compared to a control reference LSF expression level,e.g., in a normal non-cancerous sample. In some embodiments, a subjectis at risk of having a cancer if the level of LSF expression in thebiological sample from the subject is above a pre-determined referenceLSF expression threshold level. In some embodiments, the reference LSFexpression threshold level the based on the level of LSF expression anon-cancer cell or non-tumor tissue, or a control cell line, or cellsfrom a normal tissue sample, where in the tissue sample is a biologicaltissue sample from a tissue matched, and species matched and age matchedbiological sample. In some embodiments, the reference level is based ona reference sample is from a non-cancer matched tissue sample.

In some embodiments, the level of LSF expression is measured in abiological sample comprising a tumor sample. In some embodiments, abiological sample obtained from the subject comprises cancer cells, andcan be a biological sample which is serum plasma, blood or tissuesample. In some embodiments, a biological sample is selected from thegroup consisting of; a tissue sample; a tumor sample; a tumor cell; abiopsy sample; ex vivo cultivated sample; ex vivo cultivated tumorsample; surgically dissected tissue sample, blood sample, plasma sample,cancer sample, lymph fluid sample or primary ascite sample. Inalternative embodiments, the biological sample includes, for exampleblood, plasma, serum, urine, spinal fluid, plural fluid, nippleaspirates, lymph fluid, external secretions of the skin, respiratory,internal and genitoururinary tracts, bile, tears, sweat, saliva, organs,milk cells and primary ascite cells, biopsy tissue sample, a cancerbiopsy tissue sample, an in vitro or ex vivo cultivated biopsy tissuesample.

Screening for HCC:

In some embodiments, a subject amenable to treatment according to themethods as disclosed herein is screened for HCC. A convention biomarkerfor HCC is alpha-fetoproteins (AFP). Yang et al., 123 J. Cancer Res ClinOncol. 357 (1997). Individuals with elevated serum levels of AFP can bean indication of hepatocellular carcinoma. Other biomarkers for HCCinclude, but not limited to, the ones disclosed in the U.S. PatentApplication Nos.: US2009/0317844, US2010/0015605, US 2010/0120631, andInternational Patent Application Nos.: WO 2010/048304, WO 2005/0233392,and WO2008/056854, which are incorporated herein in their entirety byreference. One of the skill in the art can easily perform themeasurement of mRNA or protein level of these biomarkers in a biologicalsample e.g. blood from a subject such as human, using the standardmethods in the art. In some embodiments, a subject identified with HCCis administered a LSF inhibitor according to the methods as disclosedherein.

As disclosed herein, a subject with HCC can also be selected bydetecting a high level of LSF expression in a biological sample such asa liver sample from the subject as compared to a reference level. In oneembodiment, the reference level is the level of LSF in a normal healthysubject.

In addition, a biopsy can be used to diagnose HCC. (Walter et al., 24Curr Opin Gastroenterol. 312 (2008)). Other diagnostic methods for HCCknown to one of the skill in the art include imaging methods such asultrasound, computed tomography (CT) scan and MRI (Scholmerich et al.,52 Gut. 1224 (2004)). In various embodiments, a pharmaceuticalcomposition comprising a LSF inhibitor as disclosed herein can beadministered to a subject diagnosed with HCC or HCC susceptibility.

In some embodiments, a subject undergoing treatment of HCC, e.g.chemotherapy, can be treated alone or in combination with the methodsand compositions of the invention as disclosed herein. For example, theinventors have previously reported in collaboration with otherscientists that inhibition of LSF can increase sensitivity of HCC cellsto chemotherapeutic agents, such as, but not limited to 5-fluorouracil(5-FU) (see Yoo et al., PNAS, 2010; 107; 8357-8362). Accordingly, insome embodiments, subjects with no response to current HCC therapeutictreatment, e.g. HCC subjects who have shown chemoresistance tochemotherapeutic agents such as 5-FU, can be administered with a LSFsmall molecule inhibitor as disclosed herein using the methods andcompositions of the invention, prior to or concurrently withchemotherapy.

Detection of hepatocellular carcinoma can be difficult as most of thepatients who develop this tumor have no symptoms other than thoseinflicted with their longstanding liver disease. The onset of abdominalpain, weight loss, early satiety, jaundice and a palpable mass in theupper abdomen usually indicate an advanced cancer. Accordingly, in someembodiments, subjects at high risk for HCC can be administered aninhibitor of LSF as disclosed herein in the methods and compositions forprevention of the development of HCC (e.g. prophylactic treatment). Forexample, subjects highly susceptible to HCC are subjects with HBV, HCV,chronic alcohol consumption, an exposure to aflatoxin B1 in food (whichis a liver carcinogenic chemical produced by a mold called Aspergillusflavus after food has been stored in a hot and humid environment),hepatic adenoma resulted from the use of female hormones (estrogens) andprotein-building (anabolic) steroids, an exposure to thorotrast (apreviously used contrast agent for imaging, which caused a cancer of theblood vessels in the liver called hepatic angiosarcoma), hepaticangiosarcomas (resulted from a prolonged exposure to vinyl chloride, acompound used in the plastics industry), hereditary hemochromatosis (adisorder that causes the body to accumulate excessive amounts of iron),emphysema and cirrhosis (resulted from alpha 1 anti-trypsin deficiency)and hereditary tyrosinemia.

In additional embodiments, for prophylactic treatment (e.g. to preventreoccurrence of HCC), subjects who was diagnosed with HCC before and HCCis in remission can be selected for treatment with a LSF inhibitor asdisclosed herein using the methods and compositions of the invention.For example, subjects who had their HCC tumor removed by hepatectomyand/or liver transplantation, or who had their HCC tumor reduced orstabilized by other therapeutic methods are amenable to administrationof a LSF inhibitor or a pharmaceutical composition thereof as disclosedherein.

In yet other embodiments, subjects amenable to therapeutic treatmentwith methods and compositions of the invention, e.g. using a LSFinhibitor as disclosed herein, include subjects in need of inhibition ofLSF. For example, it has been reported that HIV patients, or individualsin need for a decrease in inflammatory response or immune function canbe treated by inhibiting LSF (Bovolenta et al, 163 J. Immuno. 6892,(1999), U.S. Patent Application No.: US2009/0081183 and InternationalApplication No.: WO1998/36641, which are incorporated herein in theirentirety by reference). Accordingly, a LSF inhibitor of the invention asdisclosed herein can be administered alone, or concurrently with otherLSF inhibitors such as IL2, or peptides, antibodies or antisense RNAagainst LSF, to subjects in which inhibition of LSF is desirable, suchas HIV.

In still another embodiment, a subject who has other cancers such asbreast cancer but has an up-regulated expression of LSF as compared to areference level can be selected for therapeutic treatment with methodsand compositions of the invention using a LSF inhibitor as disclosedherein. In some embodiments, a reference level is the expression of LSFin a normal healthy subject. In other embodiments, a reference level isthe expression of LSF in the same subject measured at the previous timepoint of the treatment regime. Other cancer indications that can be usedfor the purposes of the invention include brain cancer, colon cancer,head and neck squamous cell carcinoma, lung cancer, pancreatic cancer,ovarian cancer, and thyroid cancer.

Pharmaceutical Compositions Comprising a LSF Inhibitor of any ofCompounds (I) to (XXVI) and (III′)

Another aspect of the present invention relates to pharmaceuticalcompositions for treatment of diseases or disorders where it istherapeutically beneficial to inhibit LSF, e.g hepatocellular carcinoma.In some embodiments, a pharmaceutical composition of the inventioncomprises a therapeutically effective amount of at least one LSFinhibitor selected from any of the compounds represented by formula (I)to (XXVI) and (III′) disclosed herein. In one embodiment, a LSFinhibitor is a compound of formula (IV). In some embodiments, a LSFinhibitor is a compound selected from the group consisting of compoundsof formulae (V) to (XXVI). In some embodiments, a LSF inhibitor is thecompound FQI-34. In some embodiments, a LSF inhibitor is the compoundFQI-Br, FQI-F, FQI-Cl or FQI32. In various embodiments, a LSF inhibitoris an enantiomer, a prodrug, a derivative, or a pharmaceuticallyacceptable salt of a compound of any of formula (I) to (XXVI) and(III′).

A LSF inhibitor as disclosed herein selected from any of formula (I) to(XXVI) and (III′) can be used in an amount of about 0.001 to 10 mg/kg ofbody weight or about 0.005 to 8 mg/kg of body weight or about 0.01 to 6mg/kg of body weight or about 0.1 to 0.2 mg/kg of body weight or about 1to 2 mg/kg of body weight. In some embodiments, an inhibitor of LSF canbe used in an amount of about 0.1 to 1000 μg/kg of body weight or about1 to 100 μg/kg of body weight or about 10 to 50 μg/kg of body weight. Insome embodiments, a LSF inhibitor as disclosed herein selected from anyof formula (I) to (XXVI) and (III′) can be used at a concentration ofabout 0.001 mg/ml or 0.1 mg/ml or a higher concentration of 0.1 mg/ml.In some embodiments, a pharmaceutical composition comprises at least oneLSF inhibitor at a concentration of about 0.01 μM to 300 μM, or about0.1 μM to 150 μM, or about 1 μM to 50 μM, or about 1 μM to 25 μM. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

Collaborative data from animal studies using an in vivo mouse modelwhere a LSF inhibitor (e.g. formula IV) is injected intraperitoneallyonce every 3 days at an effective amount of about 1.0-2.0 mg/kg of bodyweight revealed that the LSF small molecule inhibitor was effective atabrogating the growth of hepatocellular carcinoma in vivo. Depending onroutes of administration, one of skill in the art can determine andadjust an effective dosage of a LSF inhibitor disclosed herein to asubject such as a human subject accordingly, by determiningpharmacokinetics and bioavailability of a LSF inhibitor and analyzingdose-response relationship specific to a LSF inhibitor in animal modelssuch as a mouse.

Toxicity and therapeutic efficacy can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compositions that exhibit large therapeutic indices, are preferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. Thetherapeutically effective dose can be determined by one of ordinaryskill in the art, e.g. using cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe therapeutic which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture by methods disclosed in Examples 8. Aneffective dose of a LSF inhibitor can be determined in an animal modelby measuring the tumor weight and tumor volume over the course oftreatment with a LSF inhibitor as compared to no treatment. In someembodiments, a dosage comprising a LSF inhibitor is considered to beeffective if the dosage inhibits or decreases the growth of tumor weightand/or tumor volume by at least about 15%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, 95%, 99% oreven 100%, as compared to a control (e.g. in the absence of a LSFinhibitor), In some embodiments, a therapeutically effective amount of aLSF inhibitor administered to a subject is dependent upon factors knownto a person of ordinary skill, including bioactivity and bioavailabilityof a LSF inhibitor (e.g. half-life and stability of a LSF inhibitor inthe body), chemical properties of a LSF inhibitor (e.g molecular weight,hydrophobicity and solubility); route and frequency of administration,time of administration (e.g. before or after a meal), and the like.Further, it will be understood that the specific dose of thepharmaceutical composition comprising a LSF inhibitor as disclosedherein to provide the therapeutic or prophylactic benefits can depend ona variety of factors including physical condition of the subject (e.g.age, gender, weight), medical history of the subject (e.g. medicationsbeing taken, other diseases or disorders) and clinical condition of thesubject (e.g. health condition, stage of the disease). The precise doseof a pharmaceutical composition comprising a LSF inhibitor can bedetermined by methods known to a skilled artisan such as pharmacologistsand physicians.

According to the invention, a LSF inhibitor as disclosed herein selectedfrom any of formula (I) to (XXVI) can be administered prophylacticallyor therapeutically to a subject prior to, simultaneously or sequentiallywith other therapeutic regimens or agents (e.g. multiple drug regimens),in a therapeutically effective amount. In some embodiments, a LSFinhibitor administered concurrently with other therapeutic agents can beadministered in the same or different compositions.

The active ingredients (e.g. a LSF inhibitor selected from any offormula (I) to (XXVI) and (III′) of the pharmaceutical compositionaccording to the invention can be administered to an individual by anyroute known to persons skilled in the art. The routes of administrationinclude intradermal, transdermal (e.g. in slow release formulations),intramuscular, intraperitoneal, intravenous, subcutaneous, oral, buccal,nasal, rectal, epidural, topical, intrathecal, rectal, intracranial,intratracheal and intrathecal and intranasal routes. Any othertherapeutically efficacious route of administration can be used, forexample absorption through epithelial or endothelial tissues or systemicadministration. In addition, a LSF inhibitor according to the inventioncan be administered together with other components of biologicallyactive agents such as pharmaceutically acceptable surfactants,excipients, carriers, diluents and vehicles.

For parenteral (e.g. intravenous, subcutaneous, intramuscular)administration, a LSF inhibitor can be formulated as a solution,suspension, emulsion or lyophilized powder in association with apharmaceutically acceptable parenteral vehicle (e.g. water, saline,dextrose solution) and additives that maintain isotonicity (e g.mannitol) or chemical stability (e.g. preservatives and buffers). Theformulation is sterilized by commonly used techniques.

In some embodiments, the route of administration is administration bysubcutaneous route. Intramuscular administration is another alternativeroute of administration. In some embodiments, a pharmaceuticalcomposition comprising a LSF inhibitor selected from any of formula (I)to (XXVI) and (III′) can be administered as a formulation adapted forsystemic delivery. In some embodiments, the compositions can beadministered as a formulation adapted for delivery to specific organs,for example but not limited to the liver. In some embodiments, apharmaceutical composition comprising a LSF inhibitor selected from anyof formula (I) to (XXVI) and (III′) as disclosed herein can beadministered as a formulation adapted for passage through theblood-brain barrier.

Alternatively, in some embodiments, a pharmaceutical composition can beincorporated in a gel, sponge, or other permeable matrix (e.g., formedas pellets or a disk) and placed in proximity to the liver endotheliumfor sustained, local release. The composition comprising a LSF inhibitorcan be administered in a single dose or in multiple doses, which areadministered at different times.

The exact route of administration as well as the optimal dosages can bedetermined by standard clinical techniques for each specific case,mainly based on the nature of the disease or disorder and on the stageof this disease. Preferably, the medicament according to the presentinvention is applied locally or systemically, in particular, orally,intravenously, parenterally, epicutaneously, subcutaneously,intrapulmonarily by inhalation or bronchoalveolar lavage,intramuscularily, intracranially, locally into intervertebral discs orother connective tissues.

As disclosed herein, a pharmaceutical composition comprising aneffective amount of at least one LSF inhibitor selected from any offormula (I) to (XXVI) and (III′) can be administered to a subject forthe therapeutic treatment or prevention (e.g. prophylactic treatment)for diseases and disorders mediated by level of LSF.

In some embodiments, a composition of the invention comprising aninhibitor of LSF of any of formula (I) to (XXVI) and (III′) as disclosedherein is formulated for treatment of cancer, e.g. hepatocellularcarcinoma. In one embodiment, an inhibitor of LSF in compositions of theinvention as disclosed herein is a compound of formula (IV), orenantiomers (compound C1-1 or C1-2 in FIG. 1), prodrugs, derivatives orpharmaceutically acceptable salts thereof. In various embodiments,pharmaceutical compositions of the invention used for inhibition of LSFin diseases or disorders, e.g. HCC, comprises a racemic mixture of a LSFinhibitor of any of formula (I) to (XXVI) and (III′).

The phrase “racemic mixture” refers to a mixture of the two enantiomersof one compound. An ideal racemic mixture is one wherein there is a50:50 mixture of both enantiomers of a compound such that the opticalrotation of the (+) enantiomers cancels out the optical rotation of the(−) enantiomers. For example, in one embodiment, a pharmaceuticalcomposition for treatment of cancer, e.g., HCC as disclosed hereincomprises compounds C1-1 and C1-2 (shown in FIG. 1), which areenantiomers of a compound of formula (IV). In some embodiments, apharmaceutical composition for treatment of cancer, e.g., HCC asdisclosed herein comprises a compound of FQI2 (Formula (V)). In someembodiments, a pharmaceutical composition for treatment of cancer, e.g.,HCC as disclosed herein comprises the compound FQI-34. In someembodiments, a pharmaceutical composition for treatment of cancer, e.g.,HCC as disclosed herein comprises the compound FQI-Br, FQI-F, FQI-Cl orFQI32.

In some embodiments, a pharmaceutical composition comprising at leastone LSF inhibitor further comprises a second therapeutic agent. In oneembodiment, the second therapeutic agent is a chemotherapeutic agentsuch as Sorafenib or 5-FU. In some embodiments, the second therapeuticagent is a second LSF inhibitor, e.g. a compound selected from any offormula (I) to (XXVI) and (III′). In some embodiments, a second LSFinhibitor is an enantiomer of a first LSF inhibitor as disclosed herein.In other embodiments, the second therapeutic agent is a therapeutic forliver diseases such as HBV, HCV and cirrhosis.

In prophylactic applications, pharmaceutical compositions (ormedicaments) comprising a LSF inhibitor can be administered to a subjectsusceptible to, or otherwise at risk of, a disease or disorder mediatedby level of LSF in an amount sufficient to eliminate or reduce the riskor delay the onset of the disease. In one embodiment, the disease ordisorder to be prevented is hepatocellular carcinoma (HCC). As most HCCsare generated from the background of hepatitis B virus (HBV) orhepatitis C virus (HCV), a subject with HBV or HCV can be subjected toan effective amount or dose of a pharmaceutical composition comprising aLSF inhibitor described herein. In one embodiment, a pharmaceuticalcomposition of the invention disclosed herein comprises a LSF inhibitorof formula (IV), or enantiomers, prodrugs, derivatives orpharmaceutically acceptable salts thereof. In some embodiments, aneffective amount or dose of a pharmaceutical composition comprising aLSF inhibitor disclosed herein can be administered to a subject at highrisk of HCC. In additional embodiments, a pharmaceutical compositionfurther comprises a second therapeutic agent, e.g. therapeutics to treathigh-risk factors such as liver diseases (e.g HBV). Representativehigh-risk factors of HCC include hepatic cirrhosis, chronic alcoholconsumption, (prolonged) exposure to liver carcinogenic chemicals suchas aflatoxin B1 in food, thorotrast in diagnostic contrast agent andvinyl chloride, hepatic adenoma, hepatic angiosarcoma, hepaticangiosarcomas, hereditary hemochromatosis, emphysema and cirrhosisresulted from alpha 1 anti-trypsin deficiency, and hereditarytyrosinemia. In various embodiments, individuals that have discontinuedtreatment for high-risk factors of HCC can still be subjected to apharmaceutical composition comprising an effective dose of LSF inhibitorselected from any of formula (I) to (XXVI) and (III′) as disclosedherein for prevention of development of HCC. For such embodiments, aneffective dose of a LSF inhibitor can be higher or lower than theprevious dosage.

In therapeutic applications, according to the invention provided herein,when an effective amount or effective dose of a pharmaceuticalcomposition comprising a LSF inhibitor selected from any of formula (I)to (XXI) and (III′) of the present invention is administered to thesubject with cancer, e.g. hepatocellular carcinoma, progression ofcancer, e.g. HCC, can be delayed or inhibited. In some embodiments,administration of an effective amount or effective dose of apharmaceutical composition comprising a LSF inhibitor selected from anyof formula (I) to (XXVI) and (III′) to a subject with hepatocellularcarcinoma can inhibit or delay progression of HCC. In furtherembodiments, treating subjects with an effective dose of apharmaceutical composition comprising a LSF inhibitor can prevent ordelay metastasis of HCC in the subject. In various embodiments, a LSFinhibitor used for therapeutic treatment of various diseases, e.g. HCC,using the methods and compositions disclosed herein is a compound offormula (IV), or enantiomers thereof or a mixture thereof. In someembodiments, a LSF inhibitor used for therapeutic treatment of variousdiseases, e.g. HCC, using the methods and compositions disclosed hereinis the compound FQI-34. In some embodiments, a LSF inhibitor used fortherapeutic treatment of various diseases, e.g. HCC, using the methodsand compositions disclosed herein is the compound FQI-BR, FQI-F, FQI-Clor FQI32. In further embodiments, a LSF inhibitor useful for suchtreatment is a compound of any of formula (I) to (XXVI) and (III′), orenantiomers, prodrugs, pharmaceutically acceptable salts thereof. Otherdiseases or disorders that can be treated by compositions of the presentinvention disclosed herein include other various cancers such as braincancer, breast cancer, colon cancer, head and neck squamous cellcarcinoma, lung cancer, pancreatic cancer, ovarian cancer, and thyroidcancer; HIV and inflammation-associated diseases.

In various embodiments, a pharmaceutical composition comprising a LSFinhibitor of the invention further comprises a second therapeutic agent.In some embodiments, a second therapeutic agent is a chemotherapeuticagent for treatment of cancer, e.g. Sorafenib. Other chemotherapeuticagents include, but not limited to, Doxorubicin, 5-FU, Paclitaxel,Irinotecan, Patupilone, Everolimus, multikinase inhibitors (e.g.Sorafenib and Sunitinib), and EGFR inhibitors (e.g. Cetuximab,Erlotinib, Gefitinib, Brivanib, Lapatinib).

In further embodiments, a second therapeutic agent is a second inhibitorof LSF. In one embodiment, the second inhibitor of LSF is a differentLSF inhibitor (e.g. IL2 or a different compound selected from formula(I) to (XXVI) and (III′). In one embodiment, the second inhibitor of LSFis an enantiomer of a first LSF inhibitor. For example, compounds C1-1and C1-2 (shown in FIG. 1) are enantiomers of the same compound offormula (IV). In such embodiments, pharmaceutical compositions of theinvention comprise a racemic mixture of the same compound. Enantiomersof the same compound can have different biological activity.Accordingly, different effective doses can be applied to enantiomers ofthe same compound in a pharmaceutical composition of the invention. Forexample, in one embodiment, a pharmaceutical composition as disclosedherein can comprise compounds C1-1 and C1-2 (enantiomers of Formula IV)at different effective amounts or doses. In alternative embodiments, aninhibitor of LSF can be formulated in a separate pharmaceuticalcomposition and administered to a subject separately or concurrentlywith a second therapeutic agent.

In some embodiments, the present invention also provides compositionsuseful for practicing the therapeutic and prophylactic methods describedherein. In some embodiments, combinations of a LSF inhibitor and anothertherapeutics can be tailored to be combined in a pharmaceuticalcomposition, where each therapeutics can target a different symptom, adifferent disease or a different disorder. In further embodiments, a LSFinhibitor and another therapeutics can be mixed together in apharmaceutical composition as disclosed herein. In other embodiments, aLSF inhibitor and another therapeutics can be present in a differentformulation when combined in a pharmaceutical composition. For example,in one embodiment, a LSF inhibitor can be present in a liquidformulation, while another therapeutics is lypholized into powder. Theformulations of different active ingredients in a pharmaceuticalcompositions as disclosed herein (e.g. a LSF inhibitor and/or anothertherapeutics) can be optimized accordingly by various factors such asphysical and chemical properties of a drug, bioavailability, route ofadministration, and whether it is a sustained or a burst release for thedrug. Therapeutic and prophylactic compositions of the present inventioncan further comprise a physiologically tolerable carrier together with aLSF inhibitor as disclosed herein, or derivatives, enantiomers, prodrugsor pharmaceutically acceptable salts thereof. In additional embodiments,a LSF inhibitor and another therapeutics can employ differentphysiologically tolerable carriers when combined in a pharmaceuticalcomposition of the invention as disclosed herein.

In some embodiments, a pharmaceutical composition as disclosed hereincomprises a LSF inhibitor together with other therapeutics and apharmaceutically acceptable excipient. Suitable carriers for a LSFinhibitor of the invention, and their formulations, are described inRemington's Pharmaceutical Sciences, 16th ed., 1980, Mack PublishingCo., edited by Oslo et al. Typically an appropriate amount of apharmaceutically acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the carrier include buffers suchas saline, Ringer's solution and dextrose solution. Further carriersinclude sustained release preparations such as semipermeable matrices ofsolid hydrophobic polymers, which matrices are in the form of shapedarticles, e.g. liposomes, films or microparticles. It will be apparentto those of skill in the art that certain carriers can be morepreferable depending upon for instance the route of administration andconcentration of a LSF inhibitor being administered.

In some embodiments, bioavailability of a LSF inhibitor according to theinvention can also be improved by using conjugation procedures whichincrease the half-life of a LSF inhibitor in a subject, for examplelinking a LSF inhibitor to polyethylene glycol, as described in WO92/13095, which is incorporated herein in its entirety by reference.

In some embodiments, bioavailability of a LSF inhibitor according to theinvention can be also enhanced by encapsulating a LSF inhibitor inbiocompatible delivery vehicles which increase the half-life of a LSFinhibitor in a human body. Exemplary biocompatible delivery vehiclesinclude polymeric vehicles such as PEG-based vehicles, or liposome-basedvehicles.

In some embodiments, a LSF inhibitor can be dissolved or dispersed as anactive ingredient in the physiologically tolerable carrier to increasethe half-life of a LSF inhibitor in a subject.

The preparation of a pharmacological composition that contains activeingredients (e.g. a LSF inhibitor) dissolved or dispersed therein iswell understood in the art and need not be limited based on formulation.Typically such compositions are prepared as injectable either as liquidsolutions or suspensions, however, solid forms suitable for solution orsuspension in liquid prior to use can also be prepared. The preparationcan also be emulsified or presented as a liposome composition. In someembodiments, a LSF inhibitor can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. In addition, if desired, the composition comprising a LSFinhibitor can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents and the like whichenhance the effectiveness of the active ingredient.

Physiologically tolerable carriers (i.e. physiologically acceptablecarriers) are well known in the art. Selection of pharmaceuticallyacceptable carriers can be accomplished by means of administration by askilled artisan. For example, if the composition is orally administered,it can be formulated in coated tablets, liquids, caplets and so forth.Exemplary of liquid carriers are sterile aqueous solutions that containno materials in addition to the active ingredients and water, or containa buffer such as sodium phosphate at physiological pH value,physiological saline or both, such as phosphate-buffered saline. Stillfurther, aqueous carriers can contain more than one buffer salt, as wellas salts such as sodium and potassium chlorides, dextrose, polyethyleneglycol and other solutes. For topical application, the carrier may be inthe form of, for example, and not by way of limitation, an ointment,cream, gel, paste, foam, aerosol, suppository, pad or gelled stick. Insome embodiments, compositions are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared. The preparation also can be emulsified or encapsulated inliposomes or micro particles such as polylactide, polyglycolide, orcopolymer for enhanced adjuvant effect, as discussed above (see Langer,Science 249, 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28,97-119 (1997). An inhibitor of LSF of this invention can be administeredin the form of a depot injection or implant preparation which can beformulated in such a manner as to permit a sustained or pulsatilerelease of the active ingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications. For suppositories, binders and carriersinclude, for example, polyalkylene glycols or triglycerides; suchsuppositories can be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%. Oralformulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

A skilled artisan will be able to determine the appropriate way ofadministering pharmaceutical compositions comprising at least one LSFinhibitor as disclosed herein in view of the general knowledge and skillin the art.

Treatment Regimes

Another aspect of the present invention relates to methods fortherapeutic and prophylactic treatment of diseases or disorders, whereinhibition of LSF is desirable. The methods comprise administering to asubject in need thereof a pharmaceutical composition comprising atherapeutically effective amount of at least one LSF inhibitor selectedfrom any of compounds represented by formula (I) to (XXVI) and (III′) asdisclosed herein.

In some embodiments, such diseases or disorders that can be treated orprevented by methods and compositions of the invention as disclosedherein include but not limited to, various cancers such as liver cancer(e.g. hepatocellular carcinoma (HCC), hepatoblastoma, cholangiocarcinoma(cancer of the bile duct), angiosarcoma), brain cancer, breast cancer,colon cancer, head and neck squamous cell carcinoma, lung cancer,pancreatic cancer, ovarian cancer, cervical cancer, melanoma and thyroidcancer; HIV; inflammation-related diseases such as hepatitis B virus(HBV), hepatitis C (HCV), cirrhosis and Alzheimer's disease; liverdiseases such as HBV, HCV, cirrhosis, hepatic adenoma, hepaticangiosarcoma and hepatic angiosarcomas; emphysema; and hereditaryhemochromatosis.

In one embodiment, hepatocellular carcinoma is treated or prevented bythe methods and compositions of the present invention with a LSFinhibitor as disclosed herein. In some embodiments, a LSF inhibitorpresent in pharmaceutical compositions of the invention is the compoundFQI-34. In some embodiments, a LSF inhibitor present in pharmaceuticalcompositions of the invention is the compound FQI-Br, FQI-F or FQI-Cl.In some embodiments, a LSF inhibitor present in pharmaceuticalcompositions of the invention is the compound FQI32. In one embodiment,a LSF inhibitor present in pharmaceutical compositions of the inventionis a compound of formula (IV), or enantiomers, prodrugs, derivatives andpharmaceutically acceptable salts thereof. In some embodiments, a LSFinhibitor is a compound selected from the group consisting of compoundsof formula (V) to (XXVI) and (III′).

Effective doses of the pharmaceutical composition comprising a LSFinhibitor as disclosed herein, for the treatment of the above describedexamples of diseases or disorders mediated by level of LSF varydepending upon many different factors, including means ofadministration, target site, physiological state of the subject, whetherthe subject is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. In one embodiment, thedisease or disorder to be treated or prevented is hepatocellularcarcinoma.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, a relatively low dosage is administered at relativelyinfrequent intervals over a long period of time. For example, subjectsat high risk of HCC, e.g. individuals with hepatitis B virus orhepatitis C virus can be treated with a LSF inhibitor as disclosedherein in a prophylactic regime to prevent the onset of HCC. Somesubjects continue to receive treatment for the rest of their lives. Insome embodiments, individuals with HCC in remission after HCC treatment(e.g. hepatectomy, liver transplantation or other therapeutic treatmentsuch as chemotherapy) can be subjected to a pharmaceutical compositioncomprising a LSF inhibitor as disclosed herein, as a preventive measureagainst recurrence of HCC. Depending on the clinical condition of asubject, dosage and frequency of pharmaceutical compositions of thepresent invention can be adjusted accordingly over time by one of theskill in the art, e.g physicians.

In therapeutic applications, a relatively high dosage in relativelyshort intervals is sometimes required until progression of the diseaseis reduced or terminated, or until the subject shows partial or completeamelioration of symptoms of disease. Thereafter, the subject can beadministered a prophylactic regime. For example, subjects with HCC canbe treated with a LSF inhibitor as disclosed herein at an effective dosein a therapeutic regimen accordingly to prevent or delay the progressionof HCC or metastasis. In other embodiments, a LSF inhibitor can beadministered using the methods and compositions as disclosed herein tochemotherapy subjects in order to increase sensitivity to chemotherapy.In some embodiments, an inhibitor of LSF as disclosed herein can beadministered to subjects prior to, concurrently with, or sequentially totreatment with chemotherapeutic drugs, e.g. Sorafenib. In furtherembodiments, HCC subjects selected for other therapeutic procedures orsurgeries, such as hepatectomy, intralesional ethanol injection, orchemoembolization, can be subjected to a treatment with a LSF inhibitoras disclosed herein. For example, a pharmaceutical composition of theinvention can be administered prior to, during or after therapeuticprocedures. Route of administration can vary with therapeutic proceduresor surgeries and can be determined by a skilled artisan. In yet anotherembodiment, compositions and methods of the invention can be used as anadjuvant therapy.

In some embodiments, the subject is a human, and in alternativeembodiments the subject is a non-human mammal. Treatment dosages need tobe titrated to optimize safety and efficacy. The amount of a LSFinhibitor depends on the stage of the disease, e.g. HCC as well aswhether a second therapeutic agent is also administered. The secondtherapeutic agent can be an agent to treat a different disease ordisorder. In some embodiments, the second therapeutic agent can be achemotherapeutic agent such as Doxorubicin, 5-FU, Paclitaxel,Irinotecan, Patupilone, Everolimus, multikinase inhibitors (Sorafeniband Sunitinib), and EGFR inhibitors (Cetuximab, Erlotinib, Gefitinib,Brivanib, Lapatinib). In alternative embodiments, the second therapeuticagent can be a second LSF inhibitor. In some embodiments, a second LSFinhibitor can be selected from the group consisting of compounds offormulae (V) to (XXVI) and (III′), and enantiomers, prodrugs andpharmaceutically acceptable salts thereof. In one embodiment, a secondLSF inhibitor can be an enantiomer of a first LSF inhibitor. In furtherembodiments, a second therapeutic agent is another therapeutics totarget another disease, or another disorder, or a different symptom. Incombination with other therapeutics, the dosage of a LSF inhibitor canbe reduced, compared to the standard dosage of a LSF inhibitor whenadministered alone.

In some embodiments, a LSF inhibitor can be administered to a subject ina pharmaceutical composition comprising an amount of a LSF inhibitor ofabout 0.001 to 10 mg/kg of body weight or about 0.005 to 8 mg/kg of bodyweight or about 0.01 to 6 mg/kg of body weight or about 0.1 to 0.2 mg/kgof body weight or about 1 to 2 mg/kg of body weight. In someembodiments, a LSF inhibitor can be used in an amount of about 0.1 to1000 μg/kg of body weight or about 1 to 100 μg/kg of body weight orabout 10 to 50 μg/kg of body weight. In some embodiments, a LSFinhibitor can be administered at a concentration of about 0.001 mg/ml or0.1 mg/ml or a higher concentration of 0.1 mg/ml. In alternativeembodiments, a pharmaceutical composition comprises at least one LSFinhibitor at a concentration of about 0.0104 to 300 μM, or about 0.1 μMto 150 μM, or about 1 μM to 50 μM, or about 1 μM to 25 μM.

The inventors in collaboration with other scientists have demonstratedthat a LSF inhibitor (e.g., a compound of formula IV or III′) reducesHCC tumor volume in vivo in a mouse model by about 80% (data not shown).Accordingly, in some embodiments, a LSF inhibitor selected from any offormula (I) to (XXVI) and (III′) can be administered to a subject (e.g.a HCC subject) according to the methods as disclosed herein in aneffective dose to reduce tumor volume by at least about 15%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 98%, about 99%, or about 100%. Tumor volumeand tumor weight in a subject can be determined by one of ordinary skillin the art, e.g. using imaging techniques such as MRI, CAT scan, and thelike.

In collaboration with other scientists, the inventors have discoveredthat in an in vivo mouse model of HCC, the administration of a smallmolecule inhibitor of LSF a compound of formula (IV) as disclosed hereininjected intraperitoneally once every 3 days at an effective amount ofabout 1.0-2.0 mg/kg prevented or delayed the growth of hepatocellularcarcinoma (HCC) (data not shown). Accordingly in some embodiments, thefrequency of administration can vary significantly from once a day, onceevery other day, once every 3 days, once weekly, once monthly to once ayear, depending on the disease of cancer (e.g., stage of cancer) such asHCC stage, and/or mode of administration.

Generally, effective dosages and dosing schedules can be adjusted basedon, for example, the outcome of the treatment such as whether theprogression rate of HCC is slower or terminated, or whether at least oneof the symptoms associated with HCC is reduced. In accordance with theteachings provided herein, the effectiveness of the treatment can bemonitored by obtaining a biological sample from a subject, e.g. a bloodserum sample, and determining the level of biomarkers for HCC, such asAFP in the serum sample, using methods well known in the art and thediagnostic methods as disclosed later herein. The efficacy of thetreatment can also be monitored by imaging modalities such as CT scan,MRI, ultrasound, and the like that are known to a skilled artisan.

In some embodiments, the daily dose administered to a subject in a formof a bolus composition comprising a LSF inhibitor can be given in asingle dose, in divided doses or in sustained release form effective toobtain the desired results. Second or subsequent administrations can beperformed at a dosage which is the same, less than or greater than theinitial or previous dose administered to the individual. A second orsubsequent administration can be administered during or prior to onsetof the disease. It is also within the skill of the art to start doses atlevels lower than required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved.

The pharmaceutical compositions comprising at least one LSF inhibitorselected from any of formula (I) to (XXVI) and (III′) as disclosedherein can be administered by parenteral, topical, intravenous, oral,subcutaneous, intraperitoneal, intranasal or intramuscular means forprophylactic and/or therapeutic treatment. For example, for treatment ofcancer, e.g., HCC, a pharmaceutical composition comprising at least oneLSF inhibitor can be injected systemically such as by intravenousinjection, or by injection or application to the relevant site, such asby direct injection into a tumor, or direct application to the site whenthe site is exposed in surgery. Other routes of administration of a LSFinhibitor as disclosed herein are intramuscular (i.m.), intravenous(i.v.), subcutaneous (s.c.), or orally, although other routes can beequally effective. Intramuscular injection is most typically performedin the arm or leg muscles. In some embodiments, pharmaceuticalcompositions as disclosed herein for the treatment ofinflammation-associated disease of the brain such as Alzheimer's diseasecan be administered directly to the head or brain via injection directlyinto the cranium. In some methods, a LSF inhibitor as disclosed hereincan be administered as a sustained release composition or device, suchas a Medipad™ device. In some methods, the sustained release compositionor device is implanted directly close to or at the site of the HCCtumor.

In some embodiments, a LSF inhibitor selected from any of formula (I) to(XXVI) and (III′) as disclosed herein can optionally be administered incombination with other agents that are at least partly effective intreatment of LSF-mediated diseases or disorders such as cancers (e.g.HCC), HIV, and inflammation-related diseases. In other embodiments, aLSF inhibitor of the invention can be administered prior to,concurrently, or after administration of another therapeutics thattargets another disease or disorder, or a different symptom. In oneembodiment, a pharmaceutical composition comprising a LSF inhibitor canbe administered alone or in combination with other therapeutic agents totreat liver diseases such as hepatitis B virus and hepatitis C virus forthe prevention of HCC development.

In some embodiments, a LSF inhibitor selected from any of formula (I) to(XXVI) and (III′) as disclosed herein can be used alone to treat cancer,e.g. hepatocellular carcinoma. In some embodiments, a LSF inhibitor canalso be administered in combination with other therapeutic procedures orsurgeries for HCC treatment such as curative hepatectomy, intralesionalethanol injection, chemoembolization, radiofrequency ablation,cryosurgery, radiation therapy, percutaneous intralesional ethanolinjection, transarterial chemoembolization, radiotherapy, systemicchemotherapy and surgery. In further embodiments, a chemotherapy subjectcan be subjected to an inhibitor of LSF using the compositions andmethods of the invention prior to, simultaneously with, or subsequentlyafter treatment with other chemotherapeutic agents, such as Doxorubicin,5-FU, Paclitaxel, Irinotecan, Patupilone, Everolimus, multikinaseinhibitors (Sorafenib and Sunitinib), and EGFR inhibitors (Cetuximab,Erlotinib, Gefitinib, Brivanib, Lapatinib). An inhibitor of LSF selectedfrom any of formula (I) to (XXVI) and (III′) can also be administeredafter surgery or after the other aforementioned treatments of cancer,e.g., where solid tumors have been removed to prevent cancer recurrenceor metastasis.

In various embodiments, a LSF inhibitor selected from any of formula (I)to (XXVI) and (III′) can be a pro-drug, where it is activated by asecond agent. Accordingly, in such embodiments, administration of suchthe second agent which activates the pro-drug into its active form canbe administered the same time, concurrent with, or prior to, or afterthe administration of the pharmaceutical composition comprising a LSFinhibitor as disclosed herein.

In some embodiments, a LSF inhibitor selected from any of formula (I) to(XXVI) and (III′) as disclosed herein is often administered aspharmaceutical compositions comprising an active therapeutic agent, i.e.a LSF inhibitor, and a variety of other pharmaceutically acceptablecomponents. See Remington's Pharmaceutical Science (15th ed., MackPublishing Company, Easton, Pa., 1980). The formulation of thecompositions depends on the intended mode of administration andtherapeutic application. The compositions can also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, non-therapeutic, non-immunogenic stabilizers and the like.However, some reagents suitable for administration to animals may notnecessarily be used in compositions for human use.

For parenteral administration, a LSF inhibitor selected from any offormula (I) to (XXVI) and (III′) as disclosed herein can be administeredas injectable dosages of a solution or suspension of the substance in aphysiologically acceptable diluent with a pharmaceutical carrier whichcan be a sterile liquid such as water oils, saline, glycerol, orethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents, surfactants, pH buffering substances and the likecan be present in compositions. Other components of pharmaceuticalcompositions are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil, and mineral oil. Ingeneral, glycols such as propylene glycol or polyethylene glycol arepreferred liquid carriers, particularly for injectable solutions.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Other mode of administration includes systemic delivery. In someembodiments, for treatment of cancer, e.g., HCC, a pharmaceuticalcomposition comprising at least one LSF inhibitor selected from any offormula (I) to (XXVI) as disclosed herein can be injected systemicallysuch as by intravenous injection, or by injection or application to therelevant site, such as by direct injection into a tumor, or directapplication to the site when the site is exposed in surgery. In someembodiments, a pharmaceutical composition of the invention can beformulated in a tablet and used orally for systemic administration. Invarious embodiments, pharmaceutical compositions of the invention canfurther comprises non-active ingredients (i.e. ingredients that have notherapeutic values for treatment of diseases, disorders or symptoms),such as physiologically acceptable carriers.

In various embodiments, modification of a LSF inhibitor selected fromany of formula (I) to (XXVI) and (III′) specifically contemplated isattachment, e.g., covalent attachment, to a polymer. In otherembodiments, a LSF inhibitor selected from any of formula (I) to (XXVI)and (III′) can be mixed with or encapsulated in a biocompatible polymer.

In another aspect, biodegradable or absorbable polymers can provideextended, often localized, release of a LSF inhibitor selected from anyof formula (I) to (XXVI) and (III′) as disclosed herein. The potentialbenefits of an increased half-life or extended release for a therapeuticagent are clear. A potential benefit of localized release is the abilityto achieve much higher localized dosages or concentrations, for greaterlengths of time, relative to broader systemic administration, with thepotential to avoid possible undesirable side effects that may occur withsystemic administration.

Bioabsorbable polymeric matrix suitable for delivery of a LSF inhibitoras disclosed herein, or variants or fragments or derivatives thereof canbe selected from a variety of synthetic bioabsorbable polymers, whichare described extensively in the literature. Such syntheticbioabsorbable, biocompatible polymers, which may release proteins overseveral weeks or months can include, for example, poly-hydroxy acids(e.g. polylactides, polyglycolides and their copolymers),polyanhydrides, polyorthoesters, segmented block copolymers ofpolyethylene glycol and polybutylene terephtalate (Polyactive™),tyrosine derivative polymers or poly(ester-amides). Suitablebioabsorbable polymers to be used in manufacturing of drug deliverymaterials and implants are discussed e.g. in U.S. Pat. Nos. 4,968,317,5,618,563 (which are incorporated herein in their entirety byreference), among others, and in “Biomedical Polymers” edited by S. W.Shalaby, Carl Hanser Verlag, Munich, Vienna, N.Y., 1994 and in manyreferences cited in the above publications. The particular bioabsorbablepolymer that should be selected will depend upon the particular patientthat is being treated.

It was previously reported in Yoo et al., (PNAS, 2010; 107; 8357-8362)that the level of LSF expression is useful to identify a subject withHCC. Accordingly, a subject amenable to treatment using the methods andcompositions as disclosed herein can be identified by measuring thelevel of LSF in a biological sample obtained from the subject and if thelevel of LSF in the biological sample from the subject is higher by astatistically significant amount relative to a reference level of LSF,the subject likely is at risk of having HCC, and accordingly, can beadministered a composition comprising at least one small moleculeinhibitor of LSF selected from any of formula (I) to (XXVI) and (III′)as disclosed herein.

A subject is identified as suffering from HCC or having a disorderedcharacterized by increased LSF expression, when the expression level ofLSF in a biological sample obtained from the subject is higher relativeto a reference level of LSF by at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 98%, about 99% or about 100%. The extent of increase inLSF expression can indicate the grades and stages of HCC (See Yoo etal., PNAS, 2010; 107; 8357-8362). Accordingly, subjects identified withHCC or having a disorder characterized by increased LSF expression canbe treated with an effective dose of a pharmaceutical composition asdisclosed herein comprising a LSF inhibitor selected from any of formula(I) to (XXVI) and (III′) as disclosed herein to inhibit or delayprogression of HCC.

In some embodiments, a biological sample is a tissue sample, e.g. aliver sample.

In some embodiments, the level of LSF in a biological sample is comparedto a reference level, or a reference biological sample, such asbiological sample obtained from an age-matched normal control (e.g. anage-matched subject not having HCC or an age-matched normal healthysubject).

In other embodiments, in order to determine the therapeutic efficacy ofthe treatment (e.g. treatment of HCC), a reference level can be thelevel of LSF expression can be measured at a previous time point fromthe same subject on a treatment regimen.

The methods of the present invention also are useful for monitoring acourse of treatment being administered to a subject. The methods can beused to monitor both therapeutic treatment on symptomatic subject andprophylactic treatment on asymptomatic subject.

A treatment administered to a subject is considered to be effective ifthe level of expression of LSF in a biological sample obtained from thesubject is decreased relative to a reference level of LSF by at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 98%, about 99% or about100%. In such embodiments, the reference level is the measurement of LSFat a previous time point from the same subject who has been administeredto the treatment regimen. Based on the outcome of treatment, the dosageand frequency of administration using the methods and compositions asdisclosed herein can be adjusted accordingly by one of skill in the art.

In one embodiment, the biological sample for analysis is a liver sample,wherein the sample comprises at least one cell. One can use anyimmunoassay to determine the level of LSF in a biological sample, suchas ELISA or immunohistochemical methods of detecting LSF which arecommonly known in the art and are encompassed for use in the presentinvention.

In some embodiments, a method of determining the presence and/or levelof LSF in a biological sample from a subject comprises performing abinding assay. Any reasonably specific binding partner can be used. Forexample, the binding partner is labeled. For example, the assay is animmunoassay, especially between LSF and an antibody that recognizes LSF,especially a labeled antibody. It can be an antibody raised against partor all of it, such as a monoclonal antibody or a polyclonal antiserum ofhigh specificity for LSF. In some embodiments, the antibodies isspecific to mammalian LSF, such as human LSF.

Thus, any anti-LSF antibody can be used in the method to determine thepresence and/or level of LSF in a biological sample, which can be usedto detect the increased or decreased level of LSF present in adiagnostic sample. Such antibodies can be raised by any of the methodswell known in the immunodiagnostics field.

In some embodiments, an immunoassay is carried out by measuring theextent of the protein/antibody interaction of the LSF/antibodyinteraction. Any known method of immunoassay may be used. A sandwichassay or ELISA is preferred. In this method, a first antibody to themarker protein is bound to the solid phase such as a well of a plasticsmicrotitre plate, and incubated with the sample and with a labeledsecond antibody specific to the protein to be assayed. Alternatively, anantibody capture assay could be used. In some embodiments, a biologicaltest sample is allowed to bind to a solid phase, and the anti-LSFprotein antibody is then added and allowed to bind. After washing awayunbound material, the amount of antibody bound to the solid phase isdetermined using a labeled second antibody against the first.

In some embodiments, a label is preferably an enzyme. The substrate forthe enzyme may be, for example, color-forming, fluorescent orchemiluminescent.

In some embodiments, a binding partner, e.g. an antibody or a ligandbinding to LSF in the binding assay is preferably a labeled specificbinding partner, but not necessarily an antibody. The binding partnerwill usually be labeled itself, but alternatively it may be detected bya secondary reaction in which a signal is generated, e.g. from anotherlabeled substance.

In some embodiments, one can use an amplified form of assay, whereby anenhanced “signal” is produced from a relatively low level of protein tobe detected. One particular form of amplified immunoassay is enhancedchemiluminescent assay. Conveniently, the antibody is labeled withhorseradish peroxidase, which participates in a chemiluminescentreaction with luminol, a peroxide substrate and a compound whichenhances the intensity and duration of the emitted light, typically4-iodophenol or 4-hydroxycinnamic acid.

In another embodiment, an amplified immunoassay can be used which isimmuno-PCR. In this technique, the antibody is covalently linked to amolecule of arbitrary DNA comprising PCR primers, whereby the DNA withthe antibody attached to it is amplified by the polymerase chainreaction. See E. R. Hendrickson et al., Nucleic Acids Research 23:522-529 (1995). The signal is read out as before.

Alternatively, in some embodiments, one method to determine the level ofLSF in a biological sample is to use a two dimensional gelelectrophoresis to yield a stained gel and the increased or decreasedlevel of the protein detected by an increased an increased or decreasedintensity of a protein-containing spot on the stained gel, compared witha corresponding control or comparative gel.

In some embodiments, methods to determine the amount of LSF in abiological sample does not necessarily require a step of comparison ofthe level of LSF with a control sample (e.g. from a normal healthysubject), but it can be carried out with reference either to a controlor a comparative sample. Thus, in relation to HCC, measuring the amountof LSF in a biological sample can be used to determine the stage ofprogression, if desired with reference to results obtained earlier fromthe same subject or by reference to standard values that are consideredtypical of the stage of the disease. In this way, the invention can beused to determine whether, for example after treatment of the subjectwith a LSF inhibitor, the disease has progressed or not. The result canlead to a prognosis of the outcome of the disease.

In some embodiments, one method to detect the presence and/or the levelof LSF in a biological sample is to perform immunohistochemical assay ona biopsy sample, such as a liver sample. The methods for detecting thepresence and/or a level of protein on a biopsy sample are well withinthe level of skill in the art. In alternative embodiments, the mRNAlevel of LSF in a biological sample is determined by quantitative PCRwith primers designed according to the nucleotide sequence of LSF. Thedesign for primers of LSF can be performed easily by one of the skill inthe art.

In various embodiments, the level of LSF can be measured and used incombination with other biomarkers for HCC such as AFP to diagnose HCC ina subject. Other biomarkers for HCC include, but not limited to, U.S.Patent No: US 2009/0317844, US Patent No.: 2010/0015605, US2010/0120631, WO 2010/048304, WO 2005/0233392, and WO2008/056854, whichare incorporated herein in their entirety by reference.

Kits

Another aspect of the present invention relates to a kit comprising oneor more LSF inhibitor of formula (I) to (XXVI) and (III′) as disclosedherein, and instructions for carrying out a method as disclosed herein.

In some embodiments, a kit can optionally additionally comprise reagentsor agents for measuring the level of LSF expression in a tumor samplefrom the subject, for example to identify a subject amenable totreatment with the methods and compositions as disclosed herein. Suchagents are well known in the art, and include without limitation,labeled antibodies that specifically bind to LSF protein and/or mRNA andthe like. In some embodiments, the labeled antibodies are fluorescentlylabeled, or labeled with magnetic beads and the like. In someembodiments, a kit as disclosed herein can further comprise at least oneor more reagents for profiling and annotating a biological sample fromthe subject in high throughput assay.

In some embodiments, the kit can further comprise instructions foradministering a composition comprising a LSF inhibitor of formula (I) to(XXVI) and (III′) to a subject in need thereof, e.g., with cancer, e.g.,HCC and instructions for doses and the like.

In addition to the above mentioned component(s), the kit can alsoinclude informational material. The informational material can bedescriptive, instructional, marketing or other material that relates tothe methods described herein and/or the use of the components for theassays, methods and systems described herein. For example, theinformational material may describe methods for enriching the metastaticcancer cell population, for characterizing a plurality of properties ofa metastatic cancer cell population.

In some embodiments, the methods and kits comprising a LSF inhibitor ofany of formula (I) to (XXVI) and (III′) as disclosed herein can beperformed by a service provider, for example, where an investigator orphysician can send the biological sample to a diagnostic laboratoryservice provider to measure the level of LSF in the biological subjectfrom the subject. In such an embodiment, after performing themeasurement of the LSF expression, the service provider can provide theinvestigator or physician a report of the characteristics of the levelof LSF expression and if report that the subject is a suitable oramenable to be treated with a LSF inhibitor of any of compounds offormula (I) to (XXVI) and (III′) according to the methods andcomposition as disclosed herein.

In alternative embodiments, a service provider can provide theinvestigator with the raw data of the level of LSF expression (proteinand/or mRNA) and leave the analysis to be performed by the investigatoror physician. In some embodiments, the report is communicated or sent tothe investigator via electronic means, e.g., uploaded on a secureweb-site, or sent via e-mail or other electronic communication means. Insome embodiments, the investigator can send the samples to the serviceprovider via any means, e.g., via mail, express mail, etc., oralternatively, the service provider can provide a service to collect thesamples from the investigator and transport them to the diagnosticlaboratories of the service provider. In some embodiments, theinvestigator can deposit the samples to be analyzed at the location ofthe service provider diagnostic laboratories. In alternativeembodiments, the service provider provides a stop-by service, where theservice provider send personnel to the laboratories of the investigatorand also provides the kits, apparatus, and reagents for performing theassays to measure the level of LSF in the subject as disclosed herein inthe investigators laboratories, and analyses the result and provides areport to the investigator of the level of LSF expression for eachsubject, and leaves the physician to make appropriate recommendations oftreatment of the subject with a composition comprising a LSF inhibitorof any formula (I) to (XXVI) and (III′) according to the methods asdisclosed herein.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose of skill in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents and otherpublications identified are expressly incorporated herein by referencefor the purpose of describing and disclosing, for example, themethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

EXAMPLES

Although any known methods, devices, and materials may be used in thepractice or testing of the invention, the methods, devices, andmaterials in this regard are described herein.

The examples presented herein relate to methods and compositionscomprising at least one LSF inhibitor selected from any of formula (I)to (XXVI) and (III′) as disclosed herein to inhibit LSF for treatment ofcancers, for example, but not limited to HCC. Throughout thisapplication, various publications are referenced. The disclosures of allof the publications and those references cited within those publicationsin their entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art towhich this invention pertains. The following examples are not intendedto limit the scope of the claims to the invention, but are ratherintended to be exemplary of certain embodiments. Any variations in theexemplified methods which occur to the skilled artisan are intended tofall within the scope of the present invention.

Materials and Methods (for Examples 1 to 3) Synthesis of FQI1(8-(2-ethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one)

To a flame-dried 250 mL round bottom flask equipped with a stir bar wasadded 5 mmol (960 mg) (E)-2-ethoxy cinnamic acid, 100 mL toluene, and7.5 mmol (1.5 mL) thionyl chloride. The reaction was heated to 85° C.and allowed to stir at this temp for 12 h and then cooled to roomtemperature. The solvent was removed under reduced pressure and theresulting crude material was then azeotroped (2×25 mL toluene) to removeexcess thionyl chloride. The resulting material was dried under highvacuum for 2 hours and made into a 1.0 M solution in CH₂Cl₂. To a 25 mLround bottom flask fitted with stir bar and flame dried under vacuum wasadded 5 mL CH₂Cl₂, 1.0 mmol (1 mL, 1.0 M solution in CH₂Cl₂) acylchloride and 1.1 mmol (151 mg) benzo[d][1,3]dioxol-5-amine. Reaction wasbrought to 50° C. and stirred at this temperature for 15 h. Reaction wascooled to room temperature, quenched by addition of 10 mL saturatedsodium bircarbonate, extracted with EtOAc (2×10 mL), dried over sodiumsulfate, filtered and concentrated in vacuo. The crude material was thendissolved in minimal CH₂Cl₂ (8 mL) and poured into 20 mL ice coldhexane. (E)-N-(Benzo[d][1,3]dioxol-5-yl)-3-(2-ethoxyphenyl) acrylamideprecipitated from solution and was isolated via filtration, washed withice cold diethyl ether (10 mL) and dried under high vacuum for 2 h toyield 215 mg of the desired acrylamide. The resulting material was usedwithout further purification. To an 8 mL microwave reaction tube wasadded 250 μmol acrylamide (80 mg) and 4 mL trifluoroacetic acid. Thereaction was heated in a CEM microwave at 85° C. and 200 W power for 30minutes. The vessel was removed from microwave reactor and allowed tocool to room temperature. The crude reaction mixture was thentransferred to a 50 mL round bottom flask and to it was added 2 mLCH₂Cl₂ and 15 mL saturated sodium bicarbonate with stirring (portionwise, 3×5 mL), followed by extraction with CH₂Cl₂ (2×10 mL). Organiclayers were separated and combined then dried over sodium sulfate. TheCH₂Cl₂ separated by filtration and concentrated under reduced pressureusing a rotary evaporator onto silica gel. The crude material was thenpurified via flash column chromatography over silica gel with 6:4hexanes:EtOAc as the eluent to yield8-(2-ethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one (54mg, 172 μmol). The material was determined to be >95% pure by ¹H NMR,¹³C NMR, and HPLC-MS analysis.

LSF-Dependent Reporter Assays.

NIH 3T3 cells were transfected with the expression construct pEF1α-LSF,a LSF expression vector^(6,20); the reporter construct pGL3B-WT4E1b, afirefly luciferase reporter vector driven by LSF¹²; phRL-TK, a controlrenilla luciferase reporter vector; and pEFGP, essentially as previouslydescribed¹². Five hours after transfection, either vehicle (DMSO) or FQIcompound was added, keeping DMSO at 0.5%. Cell extracts were harvested36 hours post-transfection and firefly and renilla luciferase activitiesmeasured via a dual luciferase assay (Promega). Five μl of extract wasmixed with 30 μl of luciferase assay reagent (Promega), and luminescencemeasured for 10 seconds using a 20/20n luminometer (Turner Biosystems).Subsequently, 30 μl of Stop & Glo reagent (Promega) was added, andluminescence also measured for 10 seconds. LSF transactivation activityis given as relative luciferase activity, the ratio of firefly torenilla activities.

Electrophoretic Mobility Shift Assays.

LSF was translated in vitro as previously described¹⁶ for use in EMSAs.Protein was preincubated with inhibitor or DMSO and then incubated with0.5 μg poly d(I-C) for 15 min at 4° C. in 40 mM KCl, 15 mM HEPES pH 7.9,1 mM EDTA, 0.5 mM DTT, 5% glycerol. Finally, radiolabelleddouble-stranded DNA (TGGCTGGTTATGGCTGGTCAGACTAG—SEQ ID NO: 5- and itscomplement) were added for a 30-min incubation at 30° C. Results werevisualized using a PhosphorImager and quantified using ImageQuantsoftware.

Cell Proliferation Assays.

Cells were plated at 3×10⁴ cells/mL, A549 and HeLa, and 1×10⁴ cells/mL,NIH/3T3, in 96-well plates and incubated for 24 h to allow for celladhesion. LSF-library compounds were dissolved in DMSO, serial dilutedin appropriate medium and added to cells in triplicate (2 plates). DMSOwas added to control wells at a final concentration of 1%. After 72 hincubation in the presence of compound, confluent cells were assayed forgrowth proliferation with CellTiter 96® AQueous One Solution CellProliferation Assay (MTS) (Promega) according to the manufacturer'sprotocol. Plates were read at 490 nm on a microplate reader, and IC₅₀values were determined as the percentage of compound-treated cell growthto control cell growth. HCC cells—one hour after plating HCC cells onto96-well plates, compound or vehicle (DMSO) was added to the desiredconcentrations, keeping DMSO at 0.5%. At indicated time points, cellviability was assayed using the CellTiter 96® Non-Radioactive CellProliferation Assay (MTT) (Promega).

TUNEL Assays.

HCC cells were plated 24 h prior to incubation with FQI1. At theindicated time points, cells were analyzed using the FragEL™ DNAfragmentation kit (Calbiochem) according to the manufacturer's protocol.Flow cytometry was performed using a FACS caliber (Becton Dickinson) andanalyzed with Cell Quest software.

Materials and Methods (for Examples 4 and 5)

All ¹H NMR, and ¹³C NMR spectra were recorded using Varian 400 (93.94kG, 1H 400 MHz) or Varian 500 (70.5 kG, ¹³C 196 MHz) spectrometers atambient temperature in CDCl₃. Chemical shifts are reported in parts permillion as follows: chemical shift, multiplicity (s=singlet, d=doublet,t=triplet, q=quartet, m=multiplet, br=broad), coupling constant, andintegration. Infrared spectra were recorded on a Nicolet Nexus 670 FT-IRESP spectrophotometer. Optical rotations were recorded on a RudolphAutopol II Polarimeterand were reported as [α]D (concentration).Analytical thin layer chromatography was performed using EMD 0.25 mmsilica gel 60-F plates. Flash column chromatography was performed onSorbent Technologies 60 Å silica gel. Chiral HPLC analysis was performedusing an Agilent 1100 series HPLC with a diode array detector.Chiralcel®OD (Chiral Technologies Inc., 24 cm×4.6 mm I.D.) column wasused. UPLC/MS analysis was done on a Waters Acquity UPLC/MS with ELSDetection. Acrylic acids and amines were purchased from Alfa Aesar orSigma Aldrich. Non-commercially available acrylic acids were synthesizedthrough saponification of respective methyl esters following knownliterature procedure¹.

Example 1

Identification of Small-Molecule LSF Inhibitors.

The role of LSF in HCC (which is reported in Yoo et al., PNAS, 2010;107; 8357-8362, which is incorporated herein in its entirety byreference) clearly demonstrates that LSF inhibitors are prime candidatesfor the treatment of hepatocellular carcinoma. Subsequent data suggestutility in other cancer indications, including but not limited tooligodendroglioma, meningioma, GBM, breast cancer, colon cancer, HNSCC,lung cancer (adrenocarcinomas and Small cell carcinomas), pancreas,ovary, thyroid, and undifferentiated cells. In particular, the inventorsdemonstrate using an in vitro cell assay that the FQI1 and FQI2compounds of Formulas (IV) and (V) respectively as disclosed herein canbe used to inhibit the growth of a variety of cancers, as demonstratedfrom the Examples (see FIGS. 9 and 10). Accordingly, in someembodiments, a composition comprising any compound of formula (I) to(XXVI) can be used to inhibit the growth of a variety of cancers in asubject, for example, a cancer selected from one or more of kidneycancer, cancers of the hematopoietic system (e.g., blood cancers such asleukemia etc.), cancers of the endometrium (e.g., cervical cancer),breast cancer, cancers of the upper digestive tract, stomach cancer,ovarian cancer, lung cancers, liver cancers (e.g., HCC), and cancers ofthe small intestine.

Without wishing to be bound by theory, the transcription factor LSF, amember of a small family of transcription factors conserved throughoutthe animal kingdom⁹, is ubiquitously expressed in mammalian tissues andcell lines¹⁰. LSF activity is tightly controlled as cells progress fromthe quiescent state into DNA replication (G0 to S)^(11,12), and it isrequired for efficient progression of cells through the G1/Stransition^(6,7). Although LSF protein levels are usually constant, withits activity modified instead by post-translational control, LSF proteinlevels were previously discussed to be highly upregulated in tumorcells, particularly in HCC cell lines^(8,13). These elevated LSF levelswere shown to promote oncogenesis in the HCC cells, leading to thesearch for specific small molecule inhibitors of this transcriptionfactor.

Until recently, transcription factors, were generally considered to beun-druggable, in particular their specific DNA-binding activities.However, such inhibitors are now being identified for an increasingnumber of transcription factors¹⁴, with some targeting oligomerizationdomains, and others directly targeting specific DNA-interactionsurfaces. LSF is predominantly dimeric in solution, but tetrameric uponinteracting specifically with DNA sites¹⁵⁻¹⁷, potentially permittinginhibition by either mode.

Herein, the inventors were provided with the results of a screen of110,000 commercially available compounds using a fluorescencepolarization assay to identify compounds that inhibit LSF-binding to DNAin vitro. Small-molecule compounds that inhibit LSF transcriptionalactivity in tissue culture cells were identified, including multiplequinolinones. This led to the inventors synthesizing quinolinones, ofwhich examples are shown in FIG. 1. The initial set of inhibitors wereadditionally screened for specificity using an electrophoretic mobilityshift assay (EMSA), testing the ability to prevent specific DNA-bindingof LSF, but not of three other transcription factors (Sp1, Oct, andE2F1). Positive compounds from the screen (personal communication of B.Andrews and L. Briggs, Sierra Sciences, Inc.) included4-aryl-3,4-dihydroquinolin-2(1H)-ones as competent LSF inhibitors.

A LSF-dependent luciferase reporter assay was used to confirm whetherthese compounds would inhibit LSF activity in a cellular context, andidentified that all hits in the initial screen were also positive. Thus,a collection of compounds based on this structure was prepared¹⁸(FIG. 1) and a subset of the compounds (Formula IV to X, and XII toXIII) were evaluated using the luciferase reporter assay in NIH 3T3cells (FIG. 6). The factor quinolinone inhibitor 1 (FQI1, (Formula IV),FIG. 1) was identified as the most active compound from this initialcollection, which has a chemical name is8-(2-ethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one.The concentration at which LSF transactivation was inhibited 50% wasapproximately 2.4 μM (FIG. 3A). Inhibition of transcriptional activationwas specific to LSF, for example, transactivation of a USF-dependentreporter by USF was unaffected by addition of FQI1 (Formula IV) (FIG.3A).

Using FQI1's structure as a starting framework, it was sought todetermine whether inhibition of LSF was unique to the racemate or if anachiral quinolinone could function as a competent inhibitor, or ifrestricting the movement of the aryl substitution altered the moleculespotency against LSF. First, FQI1 [Formula (IV)] was separated by chiralchromatography to the corresponding (R)- and (S)-enantiomers (Formula(S)-IV); and Formula (R)-IV) (FIG. 1). Assessment in the luciferasereporter assay demonstrated that the (R)-FQI1 (Formula (R)-IV) was muchless active than the racemate while (S)-FQI1 (Formula (S)-IV) wasapproximately 3-fold more active (FIG. 3A, Table 3). Without wishing tobe bound by the theory, it was reasoned that the achiralquinolin-2(1H)-one FQI2 (Formula V) can adopt similar conformations asthe chiral counterparts¹⁹ (FIG. 2B), thereby being a competentinhibitor. Indeed the activity of FQI2 (Formula V) against LSF in thetransactivation assay proved to be as effective as the (S)-enantiomer.

An unanticipated result from the LSF-dependent reporter assays was thatthe potent FQIs reproducibly stimulated LSF transcriptional activity atlow concentrations, followed by sharp inhibition. Since the DNA-bindingmoiety of LSF is tetrameric, the inventors assessed if starting atsub-saturating levels, binding of FQIs to LSF monomers results inconformational changes that enhance LSF activity. Only at saturatinglevels of FQIs is LSF activity then ablated. Modulation of LSFDNA-binding by FQI1 (Formula IV) was also observed in electrophoreticmobility shift assays in vitro (FIG. 3B, 3C). Both initial enhancementat low concentrations and inhibition at higher concentrations wereobserved. These results mirrored the concentration-dependent effects ofFQI1 (Formula IV) on LSF transactivation, although the extent ofinhibition of DNA-binding in vitro was not as pronounced as inhibitionof activity in the cell-based reporter assay. Thus, we tested effects ofFQI1 (Formula IV) on binding of LSF to an endogenous cellular targetpromoter by chromatin immunoprecipitation. Binding of an inducibleHA-tagged LSF to the promoter of POLA1, encoding the catalytic subunitof DNA polymerase α, was efficiently blocked by FQI1 (FIG. 3D).

TABLE 3 Comparison of inhibitor concentrations for cell proliferationand LSF-dependent reporter assays for FQI compounds with GI₅₀ <50 μM.IC₅₀ (μM) LSF-dependent GI₅₀ (μM) Compound reporter assat NIH 3T3 NIH3T3 FQI2 (Formula V) 0.8 0.71 ± 0.13 (S)-FQI1 (Formula (S)-IV) 0.8 1.1 ±0.4 FQI16 (Formula VIII) 2-4* 2.3 ± 0.5 FQI3 (Formula XII) 3-5* 2.6 ±0.5 FQI1 (Formula IV) 2.4 3.0 ± 0.9 FQI12 (Formula XIII) 3-4* 4.0 ± 0.8*represents values given as a range, due to the limited number ofconcenrations tested (see FIG. 6).

Example 2

Antiproliferative activities of inhibiting LSF with FQI1 and FQI2. Givenprevious discussions that LSF activity is essential for G1/Sprogression⁶′⁷, cell proliferation assays were conducted to characterizethe antiproliferative properties of inhibiting LSF with the identifiedquinolinone inhibitors. The lead compound, FQI1 (formula IV), inhibitedNIH 3T3 fibroblasts, HeLa S3 cells, and A549 cells at 50% inhibitoryconcentrations of 3.0, 0.79, and 6.3 μM (GI₅₀'s) respectively (Table 4).

TABLE 4 Growth-inhibitory activities of FQI compounds in different celllines GI₅₀ (μM) Compound NIH/3T3 HeLa A549 FQI1 (Formula IV) 3.0 ± 0.850.79 ± 0.04 6.3 ± 1.1 (R)-FQI1 (Formula (R)-IV) 45 ± 7  8.0 ± 1.0  18 ±3.2 (S)-FQI1 (Formula (S)-IV) 1.1 ± 0.41 0.30 ± 0.03 3.0 ± 1.3 FQI2(Formula V) 0.71 ± 0.13  0.40 ± 0.04 1.9 ± 0.9

Consistent with the observations in the luciferase reporter assay, the(R)-enantiomer was almost 10 times less active, whereas the(S)-enantiomer was determined to be at least twice as active againsteach cell line. Similarly, the achiral quinolinone inhibitor FQI2(Formula V) was determined to be as active as (S)-FQI1 (Formula (S)-IV)at inhibiting cell growth. Strikingly, the half maximal concentrationsfor growth inhibition of NIH 3T3 cells matched, within experimentalerror, the half maximal concentrations for inhibition of LSFtranscriptional activity (2.4 μM, 0.8 μM, and 0.8 μM), measured by theluciferase reporter assay also in NIH 3T3 cells (Table 4 and FIG. 3A).The structure activity relationships determined by the luciferasereporter assay and antiproliferative activities are consistent withthese observations. This concordance demonstrates that theantiproliferative properties of these compounds are due directly toinhibition of LSF.

To test whether the antiproliferative effects resulted from specific orbroad target specificity, GI₅₀ values from all the FQI analogs,containing combinations of peripheral structural variations, werecompared. Changes resulting from each modification were consistentoverall, in multiple cell lines. The high degree of specificity inferredfrom the remarkably concordant structure activity relationshipssuggested that the FQI antiproliferative phenotypes were a consequenceof targeting a single, or highly related, molecular target(s).Furthermore, the strong correlation between concentrations that inhibitcell proliferation and LSF transactivation indicated that the biologicaltarget is almost certainly the LSF family of transcription factors.

FQI1 (Formula IV) was evaluated as a part of the NIH Molecular LibrariesInitiative as a member of the Small Molecule Repository screened in theProbe Production Network. A search in PubChem (CID 656346) indicatedthat as of 22 Sep. 2010, FQI1 (Formula IV) had been assayed in 465assays and determined to be active in 19 of those assays. Three of these19 assays were confirmed activities in antiproliferative assaysperformed at the Burnham Center for Chemical Genomics (AID's 430, 431,620). Although these activities were confirmed, no inhibitoryconcentrations were reported, nor did FQI1 (Formula IV) lead to a probeseries. Further, these NIH studies do not teach or describe FQI1(Formula IV) or FQI2 (Formula V) as a LSF inhibitor, or can be used toinhibit cancer cell growth.

Example 3

Effect of FQI1 on Inhibition of HCC Cell Proliferation.

The effect of LSF small molecule inhibitors on growth properties of HCCcells, in which LSF overexpression is oncogenic⁸, was assessed. Two celllines were assayed: QGY-7703, an aggressive HCC cell line that expressesLSF at highly elevated levels, and HepG3, a non-tumorigenic cell linethat expresses LSF only at moderate levels⁸. These cells were used inearlier Examples in demonstrating the oncogenic properties of LSF:expression of a dominant negative mutant of LSF in QGY-7703 cells issufficient to reduce cell growth in vitro and in xenograft models, andexpression of LSF in HepG3 cells is sufficient to enhance cell growthand induce tumorigenic ability in xenografts. In cell viability (MTT)assays, both FQI1 (Formula IV) and FQI2 (Formula V) decreased overallcellular proliferation of QGY-7703 cells (FIG. 4A). Subsequent studiesperformed by the inventors in collaboration with other scientistsdemonstrated similar growth inhibition for other HCC lines with elevatedlevels of LSF (e.g., Huh7, data not shown). These data are consistantwith growth inhibition observed by the inventors in NIH 3T3, HeLa andA549 cells in Example 2 and 7. It was discovered that specifically inthe QGY-7703 cells, cell viability dropped between 24 and 48 hours ofincubation after treatment with FQI1 (Formula IV) (FIG. 4A). Thisdecrease in HCC cell viability was observable at 2 μM FQI1, and wasstatistically significant at higher concentrations of inhibitor (3 and10 μM; P<0.05). In contrast, although HepG3 cell proliferation was alsoinhibited by FQI compounds, cell viability never decreased over time.These discoveries demonstrated that the QGY-7703 cells can be “addicted”to the elevated activity of LSF, therefore leading to cell death uponLSF inhibition, and this was verified by a flow cytometric TUNEL assay,in which massive apoptosis (averaging 90%) was observed at 10 μM FQI1(Formula IV), with lower extents of DNA nicks observed per cell at 2 μM(FIG. 4B). In contrast to the aggressive QGY-7703 cells, HepG3 cellswere not TUNEL-positive under these conditions, consistent with theirnon-tumorigenic properties and lower expression levels of LSF. It waspreviously discussed that inhibition of LSF by high expression ofdominant negative LSF in murine fibroblasts or human prostate cancercells can cause apoptosis in S phase, due substantially to blockingexpression of thymidylate synthase. However, in the QGY-7703 cells,incubation with thymidine to overcome cellular dependence on thymidylatesynthase did not affect the decreased viability upon FQI treatment (FIG.7, indicating that blockage of additional LSF target genes or regulatedpathways can contribute to the cell death. Finally, the inventorsassessed whether FQI1 (Formula IV) affected normal hepatocytes. Nogrowth consequences or toxicity were observed in primary non-dividingmouse hepatocytes (FIG. 4D), demonstrating specificity of FQI1 growthconsequences to oncogenic cells. In particular, the inventors studieswith compounds of Formula (I) to (XXVI) in cell assays in vitro revealedthat there are very low or no toxic side effects of these compositionsin primary cells, in contrast to effects on multiple immortalized ortumorigenic cell lines. (data not shown).

Example 4

Use of the FQI1 Compound for Treatment of HCC in a Mouse In Vivo Model.

The inventors, in collaboration with other scientists performed in vivoexperiments in a subcutaneous xenograft model, where mice with smallQGY-7703-derived tumors were given multiple, spaced intraperitonealinjections of FQI1 (Formula IV) over a 2 week period, followed by anadditional 2-weeks prior to analysis. These collaborative data revealedthat there was a remarkable decrease in tumor growth, as measured by theendpoint tumor volumes and weights, and of vascularity, in the miceinjected with FQI1 (Formula IV), as measured by endpoint tumor volumes(data not shown; ˜9 fold), and tumor weights, and by the degrees ofvascularity (data not shown). Further, these collaborative data revealedthat the general toxicity of the FQI1 (Formula IV) compound was notevident in the inhibitor-treated animals, as assayed by lack of changesin body weight, feeding, grooming, posture, and general behavior, aswell as hematoxylin and eosin staining of liver, lung, heart, intestine,kidney, spleen and stomach tissue. When the endpoint tumors wereanalyzed by immunofluorescence, tumors from the FQI1-treated miceexpressed LSF at similar levels to those of control mice. However,expression of osteopontin, whose gene is activated directly by LSF⁸, wasabolished by FQI1 treatment, as was expression of Ki-67, indicative ofthe replicative capacity of the tumors (data not shown). Thesecollaborative results demonstrate the in vivo inactivation or inhibitionof LSF by FQI1 (Formula IV) treatment, of LSF activities as both atranscription factor and a proliferation driver. In contrast to thedramatic effects on tumor growth, no general toxicity was evident in theinhibitor-treated animals, as assayed by lack of changes in body weight,feeding, grooming, posture, and general behavior, as well as hematoxylinand eosin staining of multiple tissues (data not shown), and levels ofliver enzymes and protein in the blood (data not shown).

Hepatocellular carcinoma (HCC) is characterized by late stage diagnosisand a poor prognosis for treatment, usually consisting of surgicalresection of the tumor and chemotherapy¹⁻³. Currently, the only approvedtreatment for primary malignancies is sorafenib, a receptor tyrosinekinase and Raf inhibitor originally developed for primary kidney cancerthat is also marginally effective for HCC, increasing survival by 2-3months as a single treatment. Inhibition of oncogenic kinases usingsmall molecules has underscored the therapeutic benefit of oncogeneaddiction^(4,5), in which primary cancers are uniquely dependent on anoncogene for continued cell survival. It was previously discussed thatthe transcription factor LSF, that mediates G1/S progression in multiplecell types^(6,7), can function as an oncogene for HCC⁸. However, it doesnot teach or describe inhibition of the transcriptional activity of LSFwith small-molecule inhibitors for treatment of cancer, e.g., HCC. Theinvention herein has demonstrated that a family of small molecules thatinhibit proliferation of a number of cancer cell lines specificallytarget the DNA-binding and corresponding transcriptional activities ofLSF. The LSF inhibitors rapidly induce apoptosis in an aggressive HCCcell line in vitro and significantly inhibit tumor growth in a mousexenograft model, with no observable general toxicity.

In one embodiment, the FQI family of quinolones was identified herein astherapeutic compounds of formulas (I) to (XXVI) for inhibition of theubiquitously expressed transcription factor LSF, leading to theirantiproliferative consequences. In addition, the inventors discoveriesof the ability of these LSF inhibitors of formulas (I) to (XXVI) tocause apoptosis in aggressive HCC cells demonstrate that the excitingtherapeutic possibility of oncogene addiction to LSF in cancers, e.g.,hepatocellular carcinoma. It was previously discussed, in mousexenograft assays, that LSF was both sufficient (in HepG3 cells) andnecessary (in QGY-7703 cells) for aggressive tumor growth. In addition,in human patient HCC samples expression levels of LSF correlate withincreased stage and decreased differentiation state of disease⁸. Whilethe current therapeutic treatment options for HCC are limited, theinvention disclosed herein teaches that the FQI compounds describedherein are promising candidates for chemotherapeutic intervention andpharmaceutical development for HCC treatment. Further, the discovery ofoverexpression of LSF in cancer indications other than HCC demonstratesan oncogenic function of LSF in diverse other cancers (data not shown)Thus, without wishing to be bound by the theory, it is reasoned that thesmall-molecule LSF inhibitors disclosed herein can also be an effectivetherapeutic for treatment of various cancers.

Example 5

General Procedure for Synthesis of Isoquinolinones.

Synthesis of FQI1(8-(2-ethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one)(Formula IV)

To a flame-dried 25 mL round bottom flask equipped with stir bar andback filled with argon was added 7.5 mL dimethyl formamide, 134 mg (0.7mmol) 2-ethoxycinnamic acid, and 306 mg HATU (0.81 mmol, 1.15 equiv).Reaction was allowed to stir at room temperature for 1 hour and then toit was added 244 μL DIPEA (1.4 mmol, 2.0 equiv) and 112 mgmethylenedioxle aniline (0.735 mmol, 1.05 equiv). Reaction was allowedto stir at room temperature for 3 hours and then to it was added 25 mLEtOAc and 25 mL water. Organic layer was then separated and aqueouslayer was washed with EtOAc (2×25 mL). Organic layers were combined andwashed with brine, dried over sodium sulfate and collected viafiltration. Ethyl acetate solution was then concentrated onto silica geland purified via column chromatography with eluant 50:50 hexane:EtOAc.Fractions with desired material present as seen by UV were collected andconcentrated to give 230 mg desired acrylamide (84% yield). Material wasthen used without further purification. To an 8 mL microwave reactiontube was added 230 μmol acrylamide (70 mg) and 4 mL trifluoroaceticacid. The reaction was run under microwave conditions at 70° C. and 200W power for 30 minutes. Vessel was removed from microwave reactor andallowed to cool to room temperature. The crude reaction mixture was thentransferred to a 50 mL round bottom flask and placed in an icebath. Toit was added 2 mL CH₂Cl₂ and 15 mL saturated sodium bicarbonate withstirring (portion wise, 3×5 mL), followed by extraction with EtOAc (2×10mL). Organic layers were separated and combined then dried over sodiumsulfate. The EtOAc was separated by filtration and concentrated underreduced pressure using a rotary evaporator onto silica gel. The crudematerial was then purified via flash column chromatography over silicagel with 6:4 hexanes:EtOAc as the eluent to yield8-(2-ethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one (60mg, 193 μmop.

Spectral Data of Isoquinolinones

FQI1:8-(2-ethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula IV)

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.34 (t, J=7.03 Hz, 3H), 2.72-2.92(m, 2H), 3.95-4.04 (m, 2H), 4.53 (t, J=6.64 Hz, 1H), 5.84 (s, 2H), 6.32(s, 1H), 6.40 (s, 1H), 6.73-6.78 (m, 2H), 6.81 (d, J=8.60 Hz, 1H), 7.13(m, 1H), 8.03 (br. s., 1H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ ppm 14.88(s) 35.69 (s) 36.72 (s) 63.51 (s) 97.80 (s) 101.15 (s) 111.40 (s) 118.68(s) 120.57 (s) 128.19 (s) 128.29 (s) 129.74 (s) 131.80 (s) 143.57 (s)146.98 (s) 156.16 (s) 171.72 (s) FTIR CM⁻¹ 3208, 2982, 2895, 1677, 1482,1233, 1039. Low Res MS: Calculated Formula Weight (C₁₈H₁₇NO₄)=311.3.Observed M+1 ion=312.

FQI3:8-(2-Butoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula XII)

Compound was synthesized as described for FQI1 to give 44 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 0.95 (t, J=7.62 Hz, 3H) 1.43 (m, 2H) 1.75 (m, 2H)2.82 (dd, J=16.26, 6.89 Hz, 1H) 2.95 (dd, J=16.26, 6.89 Hz, 1H)3.91-4.07 (m, 2H) 4.57 (t, J=6.74 Hz, 1H) 5.90 (s, 2H) 6.44 (s, 1H)6.77-6.91 (m, 2H) 7.14-7.23 (m, 1H) 7.72 (br. s., 1H). ¹³C NMR (126 MHz,CHLOROFORM-d) δ ppm 13.84 (s) 19.34 (s) 31.35 (s) 35.98 (s) 36.70 (s)67.56 (s) 97.49 (s) 101.18 (s) 108.45 (s) 111.34 (s) 118.84 (s) 120.50(s) 128.24 (s) 129.51 (s) 131.60 (s) 143.59 (s) 146.97 (s) 156.29 (s)170.96 (s). FTIR CM⁻¹ 3213, 3065, 2958, 2930, 1676, 1483, 1386, 1242,1038. Low Res MS. Calculated formula weight (C₂₀H₂₁NO₄)=339.1, ObservedM+1 ion=340.

FQI4:8-(2-butoxyphenyl)-2,2-difluoro-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula XIV)

Compound was synthesized as described for FQI1 to give 41 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (500 MHz,CHLOROFORM-d) δ ppm 0.86 (d, J=14.92 Hz, 3H) 1.26-1.38 (m, 2H) 1.59-1.71(m, 2H) 2.72-3.00 (m, 2H) 3.85-3.99 (m, 2H) 4.56 (t, J=7.34 Hz, 1H) 6.58(s, 2H) 6.75-6.86 (m, 3H) 7.14-7.19 (m, 1H) 8.90 (br. s., 1H)¹³C NMR(126 MHz, CHLOROFORM-d) δ ppm 13.74 (s) 19.27 (s) 31.27 (s) 36.23 (s)36.36 (s) 67.58 (s) 97.98 (s) 109.38 (s) 111.55 (s) 120.67 (s) 121.68(s) 128.40 (s) 128.71 (s) 131.71 (s) 133.31 (s) 133.75 (s) 139.71 (s)142.97 (s) 156.28 (s) FTIR CM⁻¹ 3226, 3131, 2961, 2933, 1687, 1491,1245, 121167. Low Res MS: Calculated Formula Weight (C₂₀H₁₉F₂NO₄)=375.4.Observed M+1 ion=376.

FQI5: 4-(2-butoxyphenyl)-6-methoxy-3,4-dihydroquinolin-2(1H)-one(Formula VII)

Compound was synthesized as described for FQI1 to give 33 mg desiredisoquinolinone after flash column chromatography. ¹H NMR: (500 MHz,CHLOROFORM-d) δ ppm 0.87 (t, J=7.46 Hz, 3H) 1.36 (m, 2H) 1.68 (m, 2H)2.83 (m, 2H) 3.62 (s, 3H) 3.92 (m, 2H) 4.59 (t, J=6.97 Hz, 1H) 6.46 (d,J=2.20 Hz, 1H) 6.68 (m, 2H) 6.77 (m, 2H) 6.82 (d, J=8.07 Hz, 1H) 7.13(m, J=8.30, 6.30, 2.80 Hz, 1H) 8.15 (br. s., 1H). ¹³C NMR (126 MHz,CHLOROFORM-d) δ ppm 13.84 (s) 19.33 (s) 31.35 (s) 36.44 (s) 36.66 (s)55.51 (s) 67.58 (s) 111.36 (s) 112.83 (s) 114.14 (s) 116.12 (s) 120.53(s) 127.79 (s) 128.26 (s) 128.49 (s) 129.32 (s) 131.01 (s) 155.81 (s)156.33 (s). FTIR CM⁻¹ 3201, 3062, 2958, 2872, 1677, 1503, 1385, 1245.Low Res MS: Calculated Formula Weight (C₂₀H₂₃NO₃)=325.4. Observed M+1ion=326.

FQI6:8-(2-isobutoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula VIII)

Compound was synthesized as described for FQI1 to give 45 mg desiredisoquinolinone after flash column chromatography. ¹H NMR 500 MHz,CHLOROFORM-d) δ ppm 0.93 (dd, J=6.72, 2.08 Hz, 6H), 2.01 (m, 1H),2.72-2.94 (m, 2H), 3.62-3.75 (m, 2H), 4.51 (t, J=6.97 Hz, 1H), 5.82-5.85(m, 2H), 6.31 (s, 1H), 6.37 (s, 1H), 6.74-6.78 (m, 2H), 6.80 (d, J=8.31Hz, 1H), 7.11-7.16 (m, 1H), 7.84 (s, 1H)¹³C NMR (126 MHz, CHLOROFORM-d)δ ppm 18.34 (s) 18.39 (s) 27.36 (s) 35.11 (s) 35.68 (s) 73.21 (s) 96.57(s) 100.14 (s) 107.39 (s) 110.27 (s) 117.76 (s) 119.47 (s) 127.24 (s)127.41 (s) 128.41 (s) 130.61 (s) 142.59 (s) 145.96 (s) 155.26 (s) 170.11(s) FTIR CM⁻¹ 3213, 3118, 2961, 2910, 1679, 1483, 1240, 1037. Low ResMS: Calculated Formula Weight (C₂₀H₂₁NO₄)=339.4. Observed M+1 ion=340.

FQI7:2,2-difluoro-8-(2-isobutoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula XIX)

Compound was synthesized as described for FQI1 to give 32 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (500 MHz,CHLOROFORM-d) δ ppm 0.89 (dd, J=6.72, 4.77 Hz, 6H), 1.98 (m, 1H), 2.87(m, 2H), 3.69 (m, 2H), 4.56 (m, 1H), 6.57 (m, 2H), 6.80 (m, 2H), 6.83(d, J=8.07 Hz, 1H), 7.17 (m, 1H), 8.65 (s, 1H) ¹³C NMR (126 MHz,CHLOROFORM-d) δ ppm 18.26 (s) 18.31 (s) 27.30 (s) 35.23 (s) 35.47 (s)73.27 (s) 97.13 (s) 108.30 (s) 110.49 (s) 119.65 (s) 120.58 (s) 127.42(s) 127.46 (s) 127.70 (s) 128.66 (s) 130.69 (s) 132.36 (s) 132.73 (s)138.72 (s) 141.97 (s) 155.25 (s) 170.55 (s) FTIR CM⁻¹ 3226, 3132, 2963,2916, 1687, 1491, 1245, 1167, 1037. Low Res MS: Calculated FormulaWeight (C₂₀H₁₉F₂NO₄)=375.4. Observed M+1 ion=376.

FQI8: 4-(2-isobutoxyphenyl)-6-methoxy-3,4-dihydroquinolin-2(1H)-one(Formula XVI)

Compound was synthesized as described for FQI1 to give 45 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 0.86-0.96 (m, 6H), 2.00 (m, 1H), 2.72-2.97 (m, 2H),3.62 (d, J=1.17 Hz, 3H), 3.63-3.75 (m, 2H), 4.59 (t, J=7.03 Hz, 1H),6.46 (s, 1H), 6.68 (s, 2H), 6.76 (d, J=4.69 Hz, 2H), 6.81 (d, J=8.21 Hz,1H), 7.10-7.17 (m, 1H), 7.77 (br. s., 1H). ¹³C NMR (126 MHz,CHLOROFORM-d) δ ppm 18.33 (s) 18.37 (s) 27.35 (s) 35.62 (s) 54.51 (s)73.24 (s) 110.28 (s) 111.87 (s) 113.07 (s) 115.20 (s) 119.51 (s) 126.69(s) 127.27 (s) 127.56 (s) 128.22 (s) 130.01 (s) 154.81 (s) 155.30 (s)169.77 (s) FTIR CM⁻¹ 3050, 2957, 1682, 1504, 1246, 1031. Low Res MS:Calculated Formula Weight (C₂₀H₂₃NO₃)=325.4. Observed M+1 ion=326.

FQI9:8-(2-ethoxyphenyl)-2,2-difluoro-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula XX)

Compound was synthesized as described for FQI1 to give 55 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 1.39 (t, J=6.89 Hz, 3H) 2.84 (dd, J=16.41, 6.45 Hz,1H) 2.99 (dd, J=16.41, 7.62 Hz, 1H) 4.07 (m, 2H) 4.66 (t, J=6.89 Hz, 1H)6.62 (s, 1H) 6.68 (s, 1H) 6.88 (m, 2H) 7.23 (m, 2H) 8.50 (br. s., 1H)¹³CNMR (126 MHz, CHLOROFORM-d) δ ppm 14.81 (s) 35.94 (s) 36.28 (s) 63.56(s) 97.99 (s) 109.46 (s) 111.59 (s) 120.71 (s) 121.64 (s) 128.20 (s)128.66 (s) 128.68 (s) 133.37 (s) 139.68 (s) 142.98 (s) 156.13 (s) 171.16(s) FTIR CM⁻¹ 3215, 3062, 2922, 1684, 1490, 1243, 1044. Low Res MS.Calculated Formula Weight (C₁₈H₁₅F₂NO₄)=347. Observed M+1 ion=348.

FQI10: 4-(2-ethoxyphenyl)-6-methoxy-3,4-dihydroquinolin-2(1H)-one(Formula IX)

Compound was synthesized as described for FQI1 to give 59 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 1.40 (t, J=7.03 Hz, 3H) 2.84 (dd, J=16.70, 6.45 Hz,1H) 2.96 (dd, J=16.41, 7.03 Hz, 1H) 3.70 (s, 3H) 4.08 (m, 2H) 4.68 (t,J=6.74 Hz, 1H) 6.56 (s, 1H) 6.74 (s, 2H) 6.83 (d, J=4.10 Hz, 2H) 6.88(d, J=8.21 Hz, 1H) 7.20 (dt, J=8.42, 4.43 Hz, 1H) 7.73 (br. s., 1H). ¹³CNMR (126 MHz, CHLOROFORM-d) δ ppm 13.84 (s) 35.15 (s) 35.67 (s) 54.50(s) 62.51 (s) 110.43 (s) 111.81 (s) 113.23 (s) 114.98 (s) 119.57 (s)126.86 (s) 127.34 (s) 128.35 (s) 129.95 (s) 154.78 (s) 155.19 (s) 169.34(s). FTIR CM⁻¹ 3198, 3062, 2976, 1674, 1505, 1243, 1039. Low Res MS.Calculated Formula Weight (C₁₈H₁₉NO₃)=297.3. Observed M+1 Ion=298.

FQI11: 4-(2-ethoxyphenyl)-5,6,7-trimethoxy-3,4-dihydroquinolin-2(1H)-one(Formula XXIII)

Compound was synthesized as described for FQI1 to give 40 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 1.47 (t, J=6.89 Hz, 3H) 2.86 (m, 2H) 3.56 (s, 3H)3.80 (s, 3H) 3.87 (s, 3H) 4.10 (q, J=7.03 Hz, 2H) 4.91 (d, J=7.33 Hz,1H) 6.17 (s, 1H) 6.56 (d, J=8.20 Hz, 1H) 6.70 (t, J=7.62 Hz, 1H) 6.83(d, J=7.91 Hz, 1H) 7.12 (m, 1H) 7.72 (br. s., 1H)¹³C NMR (126 MHz,CHLOROFORM-d) ppm 14.90 (s) 30.15 (s) 36.56 (s) 56.15 (s) 60.86 (s)63.44 (s) 95.47 (s) 111.25 (s) 111.55 (s) 120.16 (s) 127.59 (s) 127.89(s) 130.26 (s) 134.08 (s) 138.27 (s) 151.42 (s) 153.31 (s) 155.96 (s)170.94 (s). FTIR CM⁻¹ 3058, 2975, 2935, 1684, 1490, 1244, 1116. Low ResMS. Calculated Formula Weight (C₂₀H₂₃NO₅)=357.4. Observed M+1 Ion=358.

FQI12:8-(2-methoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula XIII)

Compound was synthesized as described for FQI1 to give 32 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 2.82 (dd, J=16.12, 6.74 Hz, 1H) 2.91 (dd, J=16.26,6.59 Hz, 1H) 3.85 (s, 3H) 4.58 (t, J=6.59 Hz, 1H) 5.90 (s, 2H) 6.36 (s,1H) 6.44 (s, 1H) 6.87 (m, 3H) 7.23 (m, 1H) 7.70 (br. s., 1H)¹³C NMR (126MHz, CHLOROFORM-d) δ ppm 34.61 (s) 35.70 (s) 54.26 (s) 96.63 (s) 100.17(s) 107.37 (s) 109.59 (s) 117.67 (s) 119.74 (s) 127.20 (s) 128.55 (s)130.69 (s) 142.58 (s) 146.00 (s) 155.77 (s) 170.19 (s). FTIR CM⁻¹ 3213,2977, 2930, 1684, 1506, 1242, 1036. Low Res MS. Calculated FormulaWeight (C₁₇H₁₅NO₄)=297.3. Observed M+1 Ion=298.

FQI13:2,2-difluoro-8-(2-methoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula XXI)

Compound was synthesized as described for FQI1 to give 41 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 2.83 (dd, J=16.41, 6.15 Hz, 1H) 2.93 (dd, J=16.26,7.18 Hz, 1H) 3.70 (s, 3H) 3.85 (s, 3H) 4.67 (t, J=6.59 Hz, 1H) 6.54 (s,1H) 6.74 (s, 1H) 6.84 (m, 2H) 6.90 (d, J=8.20 Hz, 1H) 7.22 (dt, J=5.27,2.93 Hz, 1H) 7.76 (br. s., 1H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ ppm35.87 (s) 36.28 (s) 55.31 (s) 98.02 (s) 109.40 (s) 110.82 (s) 120.92 (s)121.56 (s) 128.16 (s) 128.73 (s) 131.71 (s) 133.40 (s) 139.70 (s) 143.01(s) 156.77 (s) 171.11 (s). FTIR CM⁻¹ 3235, 3025, 2982, 2922, 1684, 1489,1244, 1166. Low Res MS. Calculated Formula Weight (C₁₇H₁₃F₂NO₄)=333.Observed M+1 Ion=334.

FQI14: 6-methoxy-4-(2-methoxyphenyl)-3,4-dihydroquinolin-2(1H)-one(Formula XI)

Compound was synthesized as described for FQI1 to give 48 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 2.85 (d, J=4.69 Hz, 2H) 3.57 (s, 3H) 3.80 (s, 3H)4.89 (t, J=4.69 Hz, 1H) 6.56 (s, 1H) 6.74 (s, 2H) 6.83 (d, J=4.10 Hz,2H) 6.88 (d, J=8.21 Hz, 1H) 7.20 (dt, J=8.42, 4.43 Hz, 1H) 7.73 (br. s.,1H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ ppm 35.15 (s) 35.67 (s) 54.50 (s)62.51 (s) 110.43 (s) 111.81 (s) 113.23 (s) 114.98 (s) 119.57 (s) 126.86(s) 127.34 (s) 128.35 (s) 129.95 (s) 154.78 (s) 155.19 (s) 169.34 (s).FTIR CM⁻¹ 3255, 3044, 2934, 2915, 1683, 1506, 1242, 1110. Low Res MS.Calculated Formula Weight (C₁₇H₁₇NO₃) 283.3. Observed M+1 Ion=284.

FQI15:5,6,7-trimethoxy-4-(2-methoxyphenyl)-3,4-dihydroquinolin-2(1H)-one(Formula XXIV)

Compound was synthesized as described for FQI1 to give 35 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 2.85 (d, J=4.69 Hz, 2H), 3.57 (s, 3H), 3.80 (s, 3H),3.87 (s, 3H), 3.89 (s, 3H), 4.89 (t, J=4.69 Hz, 1H), 6.18 (s, 1H), 6.58(d, J=7.62 Hz, 1H), 6.72 (t, J=7.62 Hz, 1H), 6.86 (d, J=7.91 Hz, 1H),7.15 (t, J=7.62 Hz, 1H), 7.98 (br. s., 1H)¹³C NMR (126 MHz,CHLOROFORM-d) δ ppm 30.08 (s), 36.61 (s), 55.27 (s), 56.15 (s), 60.84(s), 60.95 (s), 95.53 (s), 110.43 (s), 111.39 (s), 120.37 (s), 127.63(s), 127.95 (s), 130.30 (s), 134.10 (s), 138.24 (s), 151.39 (s), 153.34(s), 156.59 (s), 171.04 (s). FTIR CM⁻¹ 3058, 2975, 2935, 1684, 1490,1244, 1116 Low Res MS. Calculated Formula Weight (C₁₉H₂₁NO₅)=343.4.Observed M+1 ion=344.

FQI16:8-(2-propoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula VI)

Compound was synthesized as described for FQI1 to give 41 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 0.94 (t, J=7.42 Hz, 3H), 1.74 (sxt, J=6.96 Hz, 2H),2.72-2.93 (m, 2H), 3.80-3.96 (m, 2H), 4.53 (t, J=6.64 Hz, 1H), 5.84 (s,2H), 6.31 (d, J=2.34 Hz, 1H), 6.39 (d, J=1.56 Hz, 1H), 6.73-6.79 (m,2H), 6.81 (d, J=8.60 Hz, 1H), 7.08-7.17 (m, 1H), 7.83 (br. s., 1H). ¹³CNMR (126 MHz, CHLOROFORM-d) δ ppm 9.70 (s), 21.63 (s), 34.91 (s), 35.69(s), 68.40 (s), 96.55 (s), 100.15 (s), 107.41 (s), 110.33 (s), 117.78(s), 119.49 (s), 127.22 (s), 127.32 (s), 128.53 (s), 130.62 (s), 142.57(s), 145.96 (s), 155.25 (s), 170.12 (s). FTIR CM⁻¹ 3211, 3116, 2966,2878, 1678, 1483, 1239, 1038. Low Res MS. Calculated Formula Weight(C19H19NO4)=325.4. Observed M+1 ion=326.

FQI17:2,2-difluoro-8-(2-propoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula XV)

Compound was synthesized as described for FQI1 to give 45 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (500 MHz,CHLOROFORM-d) δ ppm 0.91 (t, J=7.34 Hz, 3H), 1.67-1.76 (m, 2H),2.74-2.98 (m, 1H), 3.82-3.96 (m, 2H), 4.58 (t, J=7.09 Hz, 1H), 6.54 (s,1H), 6.60 (s, 1H), 6.78-6.82 (m, 2H), 6.84 (d, J=8.07 Hz, 1H), 7.15-7.19(m, 1H), 8.27 (s, 1H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ ppm 10.64 (s)22.59 (s) 36.22 (s) 36.26 (s) 69.48 (s) 98.11 (s) 109.37 (s) 111.56 (s)120.68 (s) 121.61 (s) 128.34 (s) 128.58 (s) 128.68 (s) 131.71 (s) 133.39(s) 133.75 (s) 139.70 (s) 142.98 (s) 156.24 (s) 171.48 (s). FTIR CM⁻¹3226, 3131, 2969, 2934, 1688, 1491, 1246, 1167. Low Res MS: CalculatedFormula Weight (C₁₉H₁₇F₂NO₄)=361.3. Observed M+1 ion=362.

FQI18: 6-methoxy-4-(2-propoxyphenyl)-3,4-dihydroquinolin-2(1H)-one(Formula X)

Compound was synthesized as described for FQI1 to give 43 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (500 MHz,CHLOROFORM-d) δ ppm 0.92 (t, J=7.46 Hz, 3H), 1.73 (m, J=7.00, 7.00,7.00, 7.00 Hz, 2H), 2.73-2.97 (m, 2H), 3.62 (s, 3H), 3.82-3.95 (m, 2H),4.60 (t, J=6.85 Hz, 1H), 6.47 (d, J=1.96 Hz, 1H), 6.63-6.72 (m, 2H),6.74-6.79 (m, 2H), 6.82 (d, J=8.07 Hz, 1H), 7.10-7.16 (m, 1H), 8.08 (br.s., 1H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ ppm 10.70 (s), 22.65 (s),36.40 (s), 36.66 (s), 55.52 (s), 69.44 (s), 111.38 (s), 112.86 (s),114.15 (s), 116.13 (s), 120.54 (s), 127.78 (s), 128.26 (s), 128.47 (s),129.33 (s), 131.01 (s), 155.81 (s), 156.31 (s). FTIR CM⁻¹ 3197, 3061,2965, 2934, 1675, 1503, 1245, 1043. Low Res MS: Calculated FormulaWeight (C₁₉H₂₁NO₃)=311.4.

FQI19:8-(4-methoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula XVIII)

Compound was synthesized as described for FQI1 to give 53 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 2.76-2.90 (m, 13H), 3.79 (s, 3H), 4.09-4.16 (m, 1H),5.90 (s, 2H), 6.37 (d, J=7.91 Hz, 2H), 6.86 (d, J=8.50 Hz, 2H), 7.09 (d,J=8.50 Hz, 2H), 7.86 (s, 1H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ ppm38.64 (s), 41.18 (s), 55.29 (s), 97.67 (s), 101.26 (s), 108.42 (s),114.30 (s), 119.68 (s), 128.70 (s), 131.14 (s), 133.51 (s), 143.59 (s),147.09 (s), 158.72 (s), 170.53 (s) FTIR CM⁻¹ 2918, 1638, 1481, 1252,1036. Low Res MS: Calculated Formula Weight (C₁₇H₁₅NO₄)=297.3. ObservedM+1 ion=298.

FQI20:2,2-difluoro-8-(4-methoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(Formula XXII)

Compound was synthesized as described for FQI1 to give 41 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 2.78-2.93 (m, 2H), 3.80 (s, 3H), 4.19 (t, J=7.77 Hz,1H), 6.59 (s, 1H), 6.66 (s, 1H), 6.88 (d, J=8.50 Hz, 2H), 7.09 (d,J=8.79 Hz, 2H), 9.02 (br. s., 1H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ ppm37.21 (s), 40.15 (s), 54.27 (s), 97.38 (s), 108.36 (s), 113.50 (s),121.36 (s), 127.70 (s), 128.66 (s), 130.69 (s), 131.54 (s), 132.13 (s),132.73 (s), 138.67 (s), 142.08 (s), 157.97 (s), 170.38 (s). FTIR CM⁻¹3228, 3002, 2911, 1685, 1490, 1251, 1168. Low Res MS: Calculated FormulaWeight (C₁₇H₁₃F₂NO₄)=333.3. Observed M+1 ion=334.

FQI21: 6-methoxy-4-(4-methoxyphenyl)-3,4-dihydroquinolin-2(1H)-one(Formula XVII)

Compound was synthesized as described for FQI1 to give 41 mg desiredisoquinolinone after flash column chromatography. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 2.83-2.91 (m, 2H), 3.69 (s, 3H), 3.79 (s, 3H), 4.20(t, J=7.47 Hz, 1H), 6.47 (s, 1H), 6.69-6.79 (m, 2H), 6.86 (d, J=8.50 Hz,2H), 7.10 (d, J=8.50 Hz, 2H), 8.30 (br. s., 1H). ¹³C NMR (126 MHz,CHLOROFORM-d) δ ppm 37.36 (s), 40.37 (s), 54.13 (s), 54.38 (s), 111.57(s), 113.15 (s), 115.42 (s), 127.39 (s), 127.67 (s), 129.45 (s), 132.12(s), 154.64 (s), 157.56 (s), 169.70 (s). FTIR CM⁻¹ 3204, 3063, 2957,2835, 1678, 1505, 1248, 1036. Low Res MS: Calculated Formula Weight(C₁₇H₁₇NO₃)=283.3. Observed M+1 ion=284.

Example 6

Synthesis of FQI2 (Formula V).

FQI2: 8-(2-ethoxyphenyl)-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one (FormulaV)

To a flame-dried 250 mL round bottom flask equipped with a stir bar wasadded 5 mmol (950 mg) 3-(2-ethoxyphenyl)propiolic acid, 100 mL toluene,and 7.5 mmol (1.5 mL) thionyl chloride. The reaction was heated to 85°C. and allowed to stir at this temp for 12 h and then cooled to roomtemperature. The solvent was removed under reduced pressure and theresulting crude material was then azeotroped (2×25 mL toluene) to removeexcess thionyl chloride. The resulting material was dried under highvacuum for 2 hours and made into a 1.0 M solution in CH₂Cl₂. To a 25 mLround bottom flask fitted with stir bar and flame dried under vacuum wasadded 5 mL CH₂Cl₂, 1.0 mmol (1 mL, 1.0 M solution in CH₂Cl₂) acylchloride and 1.1 mmol (151 mg) benzo[d][1,3]dioxol-5-amine. Reaction wasbrought to 50° C. and stirred at this temperature for 15 h. Reaction wascooled to room temperature, quenched by addition of 10 mL saturatedsodium bircarbonate, extracted with EtOAc (2×10 mL), dried over sodiumsulfate, filtered and concentrated in vaccuo. Crude material was thendissolved in minimal CH₂Cl₂ (5 mL) and poured into 20 mL ice coldhexane. N-(benzo[d][1,3]dioxol-5-yl)-3-(2-ethoxyphenyl)propiolamideprecipitated from solution and was isolated via filtration, washed withice cold diethyl ether (10 mL) and dried under high vacuum for 2 h toyield 309 mg of the desired propargyl amide. The resulting material wasused without further purification. To a flame dried 10 mL round bottomflask fitted with stir bar was added 84 mg propargyl amide (0.270 mmol,1 equiv), 500 uL CH₂Cl₂, 1 mL trifluoroacetic acid, and 3 mg Pd(OAc)₂(0.01 mmol, 0.05 equiv). Reaction was allowed to stir at roomtemperature 2 hours and then quenched by addition of 10 mL saturatedammonium chloride, extracted with CH₂Cl₂ (3×10 mL), dried over sodiumsulfate, CH₂Cl₂ isolated via filtration, concentrated onto silica geland isolated via flash column chromatography over silica gel with 7:3hexanes:EtOAc as the eluent to yield8-(2-ethoxyphenyl)-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one. Residual Pdwas then scavenged by treating with MP-TMT resin (15 mg) in 5 mL THF for24 hours at room temperature. Material was then filtered through a padof celite, and celite washed with CH₂Cl₂ (2×10 mL). CH₂Cl₂ solution wasthen concentrated onto silica gel and purified via a second silica gelcolumn with eluent 7:3 hexanes:EtOAc to give purified8-(2-ethoxyphenyl)-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one (65 mg, 232μmol). The material was determined to be >95% pure by ¹H NMR, ¹³C NMR,and HPLC-MS.

FQI2: 8-(2-ethoxyphenyl)-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one (FormulaV)

1H NMR (500 MHz, CHLOROFORM-d) δ ppm 1.12 (t, J=6.97 Hz, 3H), 3.95 (m,2H), 5.91 (d, J=1.22 Hz, 2H), 6.47 (s, 1H), 6.55 (s, 1H), 6.90 (s, 1H),6.95 (d, J=8.56 Hz, 1H), 6.99 (m, 1H), 7.15 (d, J=7.34 Hz, 1H), 7.35 (m,1H), 12.98 (br. s., 1H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ ppm 14.60(s), 63.96 (s), 96.33 (s), 101.62 (s), 104.48 (s), 112.29 (s), 114.87(s), 118.72 (s), 120.69 (s), 126.86 (s), 130.08 (s), 130.48 (s), 135.55(s), 144.13 (s), 150.43 (s), 150.99 (s), 155.71 (s), 164.40 (s). FTIRCM⁻¹ 2978, 2886, 1653, 1487, 1251, 1037. Low Res MS: Calculated FormulaWeight (C₁₈H₁₅NO₄)=309.3. Observed M+1 ion=310.

Prior to running in assays, material was digested in 10 mL of a 4.5%HNO₃ and 0.5% HF aqueous solution then analyzed via ICP-MS for Pdconcentration (6.8 ppm observed).

Chiral HPLC Separation of FQI1 to (R)-FQI1 and (S)-FQI1

To a 1.5 mL HPLC vial was added 10 mg of FQI1 and 1 mL of an 8:1:1mixture of Hexane:Isopropanol:Chloroform. Solution was sonicated for 30minutes to ensure complete dissolution. An Agilent 1100 Series HPLCfitted with analytical ChiralCel OD column (10μ particle size, 4.6 mmID, 250 mm length) was primed for 30 minutes with a 20% isopropanol inHexane mixture at a flow rate of 1 mL/min. After priming, 1004 of theFQI1 solution was injected onto the column and allowed to separate.Starting from the 9 minute mark, a number of collections were made fromthe end of the column into a series of 1.5 mL HPLC vials every 15seconds (24 vials in all with ˜250 μL collected in each vial). After the15 minute mark, the vials were diluted to 750 μL with a 20% isopropanolin hexane mixture and analyzed for enantiopurity. All vials containingmaterial with an er of >95:5 were collected and combined. This procedurewas repeated 20 times and produced 7 mg of (S)-FQI1 (retention time of9.6 minutes, [α]_(D) ²³=−8.5° (c=0.47, CHCl₃) er>99:1) and 10 mg of(R)-FQI1 (retention time of 10.6 minutes, [α]_(D) ²³=+10.5 (c=0.67,CHCl₃) er>99:1).

(S)-FQI1. Retention time 9.6 minutes. (R)-FQI1. Retention time 10.6minutes.

Optical Rotation and Determination of Absolute Stereochemistry of(S)-FQI1 and (R)-FQ11 (S)-FQI1 (Formula (S)-IV)

(−)-(S)-FQI1 [α]_(D) ²³=−8.5° (c=0.47, CHCl₃, er=99:1)

Compounds known in the literature and used for comparative analysis ofoptical rotation. (Paquin, J. F., Stephenson, C. R., Defieber, C. &Carreira, E. M. Catalytic asymmetric synthesis with Rh-diene complexes:1,4-addition of arylboronic acids to unsaturated esters. Org. Lett. 7,3821-3824 (2005); Chen, G., Tokunaga, N. & Hayashi, T. Rhodium-catalyzedasymmetric 1,4-addition of arylboronic acids to coumarins: asymmetricsynthesis of (R)-tolterodine. Org. Lett. 7, 2285-2288 (2005)

a b

[α]_(D) ²³=−53.4° [α]D²³=−45.2°

(R)-FQI1 (Formula (R)-IV)

(+)-(R)-FQI1 [α]_(D) ²³=+10.5 (c=0.67, CHCl₃, er=99:1)

Compounds known from the literature and used for comparative analysis ofoptical rotation. (Defieber, C., Paquin, J. F., Serna, S. & Carreira, E.M. Chiral [2.2.2] dienes as ligands for Rh(I) in conjugate additions ofboronic acids to a wide range of acceptors. Org. Lett. 6, 3873-3876(2004); Alper, H. & Dong, C. Enantioselective cyclocarbonylation of2-vinylanilines to six-membered lactams. Tetrahedron: Asymmetry 15,35-40 (2004).)

c d

[α]_(D)23=+37.4 [α]_(D) ²³=+21.6

Example 7

Cell Growth Inhibition Assays. FIGS. 8A-8Q show the growth inhibition(as % control) versus concentrations of different FQI (log₁₀) wasplotted to determine GI₅₀ (IC₅₀) values of inhibitors. All data arereported in micromolar (04) concentrations.

For FQI1 (Formula IV), growth inhibition of NIH/3T3 Cell LineIC50=3.0±0.85, HeLa Cell Line IC50=0.79±0.035; A549 Cell LineIC50=6.3±1.1 (FIG. 8A). For (S)-FQI1, growth inhibition of NIH/3T3 CellLine IC50=1.1±0.45, HeLa Cell Line IC50=0.3±0.025; A549 Cell LineIC50=3.0±1.3 (FIG. 8B). For (R)-FQI1 (Formula (R)-IV), growth inhibitionof NIH/3T3 Cell Line IC50=45±6.9, HeLa Cell Line IC50=8.0±1.0; A549 CellLine IC50=18±3.2 (FIG. 8C).

The growth inhibition for other compounds as shown in FIGS. 8A-8Q.Growth curve data is not shown for FQI9, where the growth inhibition ofNIH/3T3 Cell Line IC50=30±3.5, HeLa Cell Line IC50=6.6±1.2; A549 CellLine IC50=>300. Growth curve data is not shown for FQI11 (FormulaXXIII), where the growth inhibition of NIH/3T3 Cell Line IC50=>200, HeLaCell Line IC50=>200; A549 Cell Line IC50=>200. Growth curve data is notshown for FQI13 (Formula XXI), where the growth inhibition of NIH/3T3Cell Line NIH/3T3 Cell Line IC50=72±9.8, HeLa Cell Line IC50=8.8±1.4;A549 Cell Line IC50=>300. Growth curve data is not shown for FQI14(Formula XI), where the growth inhibition of the A549 Cell LineIC50=>10004. Growth curve data is not shown for FQI15 (Formula XXIV),where the growth inhibition of NIH/3T3 Cell Line NIH/3T3 Cell LineIC50=>200, HeLa Cell Line IC50=>200; A549 Cell Line IC50=>200. Growthcurve data is not shown for FQI19 (Formula XX), where the growthinhibition of NIH/3T3 Cell Line NIH/3T3 Cell Line IC50=>200, HeLa CellLine IC50=>200; A549 Cell Line IC50=>200. Growth curve data is not shownfor FQI21 (Formula XVII), where the growth inhibition of NIH/3T3 CellLine NIH/3T3 Cell Line IC50=>200, HeLa Cell Line IC50=67±9.9; A549 CellLine IC50=>200.

Example 8: Synthesis and Characterization of FQI-34 Synthesis of4-(dimethylamino)-2-ethoxybenzaldehyde

4-(dimethylamino)-2-ethoxybenzaldehyde

In a flame dried 500 mL round bottom flask was prepared a solution of4-(dimethylamino)-2-hydroxy-benzaldehyde (3.00 g, 18.16 mmol) in Acetone(181.60 mL) followed by addition of bromoethane (9.89 g, 90.80 mmol,6.78 mL), anhydrous potassium carbonate (3.76 g, 27.24 mmol) and18-crown-6 (96.01 mg, 363.20 umol). The reaction mixture was fitted witha reflux condenser and heated at 40 C for 16 hr, and then cooled to roomtemperature. The reaction was filtered and filtered solid washed withacetone. The filtrate was evaporated to dryness to give4-(dimethylamino)-2-ethoxy-benzaldehyde (3.51 g, reddish solid) inquantitative yield.

¹H NMR (400 MHz, Chloroform-d) δ 10.20 (s, 1H), 7.73 (d, J=9.0 Hz, 1H),6.29 (d, J=9.0 Hz, 1H), 6.02 (s, 2H), 4.12 (q, J=7.0 Hz, 2H), 3.07 (s,3H), 1.47 (t, J=7.0 Hz, 3H). ¹³C NMR (126 MHz, Chloroform-d) δ 187.60,163.45, 155.99, 129.92, 114.65, 104.53, 93.76, 70.10, 63.79, 40.21,14.72. IR (KBr): v (max) 2979, 2905, 2763, 1659, 1587, 1553, 1526, 1440,1372, 1360, 1287, 1241, 1108, 806 cm⁻¹. Melting point: 59° C. UV: λ(max) 351.888 nm. MS (UPLC): Calcd requires m/z [M+H]⁺194.110; found193.802.

Synthesis of(2-(benzo[d][1,3]dioxol-5-ylamino)-2-oxoethyl)triphenylphosphoniumbromide

(2-(benzo[d][1,3]dioxol-5-ylamino)-2-oxoethyl)triphenylphosphoniumbromide

A solution of triphenylphosphine (20.70 g, 78.93 mmol) dissolved inToluene (250.57 mL) was prepared in a 500 mL RBF. To the solution wasadded N-(1,3-benzodioxol-5-yl)-2-bromo-acetamide (19.40 g, 75.17 mmol)in one portion. The resulting slurry was heated to reflux and stirredfor 16 h. The resulting grey precipitate was filtered, rinsed withtoluene, and dried in vacuo to afford(2-(benzo[d][1,3]dioxol-5-ylamino)-2-oxoethyl)triphenylphosphoniumbromide (29.80 g, 57.27 mmol, 76.19% yield) as a grey powder. Crudeproduct was used in subsequent steps without further purification.

¹H NMR (500 MHz, DMSO-d6) δ 11.01 (s, 1H), 7.89-7.78 (overlap, 9H),7.77-7.70 (overlap, 6H), 7.06 (d, J=2.0 Hz, 1H), 6.84 (dd, J=8.4, 2.0Hz, 1H), 6.79 (d, J=8.4 Hz, 1H), 5.95 (s, 2H), 5.30 (d, J=14.9 Hz, 2H).13C NMR (126 MHz, DMSO-d6) δ 161.16, 147.03, 143.68, 134.93, 133.88,133.79, 132.16, 130.08, 129.98, 119.01, 118.31, 112.52, 108.08, 101.45,101.18. IR (KBr): (max) 3155, 2907, 1665, 1486, 1502, 1436, 1245, 1114,1035, 929, 737, 690 cm⁻¹. Melting Point: 225° C. UV: λ (max) 219.888. MS(UPLC): Calcd requires m/z [M−Br⁻]⁺440.141; found 440.124.

Synthesis ofN-(benzo[d][1,3]dioxol-5-yl)-3-(4-(dimethylamino)-2-ethoxyphenyl)acrylamide

N-(benzo[d][1,3]dioxol-5-yl)-3-(4-(dimethylamino)-2-ethoxyphenyl)acrylamide

In an 250 mL flask equipped with a stirbar and fitted with an argonballoon, Butyllithium (1.6 M, 13.58 mL) was slowly added to a mixture of(2-(benzo[d][1,3]dioxol-5-ylamino)-2-oxoethyl)triphenylphosphoniumbromide (10.77 g, 20.70 mmol) in Tetrahydrofuran (69.00 mL). Theresulting slurry was stirred for 30 minutes during which time themixture was slowly warmed to room temperature.4-(dimethylamino)-2-ethoxy-benzaldehyde (2.00 g, 10.35 mmol) was addedas a single portion, and the reaction was fitted with condenser andstirred at reflux for 16 h. The reaction mixture was quenched byaddition of saturated aq. ammonium chloride (150 mL), and extracted 3×50mL DCM. The combined organics were washed with brine, dried over sodiumsulfate, and concentrated in vacuo. The residue was dissolved in aminimal amount of DCM, and rinsed through a short silica plug to affordN-(benzo[d][1,3]dioxol-5-yl)-3-(4-(dimethylamino)-2-ethoxyphenyl)acrylamide(3.57 g, 10.07 mmol, 97.33% yield) as a mixture of E/Z isomers (˜3:1).Notes: yellow solid.

¹H NMR (500 MHz, Chloroform-d) δ 7.93 (d, J=15.4 Hz, 1H), 7.36 (d, J=8.8Hz, 1H), 7.20 (s, 1H), 6.86 (d, J=8.3 Hz, 1H), 6.74 (d, J=8.2 Hz, 1H),6.45 (d, J=15.4 Hz, 1H), 6.27 (dd, J=8.7, 2.4 Hz, 1H), 6.14 (d, J=2.4Hz, 1H), 5.94 (s, 2H), 4.09 (q, J=6.8 Hz, 2H), 3.00 (s, 6H), 1.48 (d,J=6.8 Hz, 3H). ¹³C NMR (126 MHz, cdcl₃) δ 165.71, 159.43, 158.29,152.87, 147.77, 138.22, 133.79, 133.19, 131.42, 130.52, 115.91, 112.95,112.27, 108.07, 104.72, 102.77, 101.22, 95.63, 77.41, 63.86, 40.33,14.97. IR (KBr): v (max) 3267, 2898, 1650, 1591, 1549, 1488, 1352, 1240,1196, 1039, 806 cm⁻¹. Melting Point: 107° C. UV: λ (max) 371.888. MS(UPLC): calc'd requires m/z [M+H]⁺: 355.166; found: 355.211.

Synthesis of8-(4-(dimethylamino)-2-ethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]quinolin-6(5H)-one(FQI-34)

8-(4-(dimethylamino)-2-ethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-]quinolin-6(5H)-one(FQI-34)

In a 250 mL round bottom flask,N-(1,3-benzodioxol-5-yl)-3-[4-(dimethylamino)-2-ethoxy-phenyl]prop-2-enamide(2.51 g, 7.08 mmol) was dissolved in trifluoroacetic acid (70.50 mL) andallowed to stir at room temperature for 2 days. Upon completion, thereaction was cooled to 0° C., and slowly neutralized by addition ofsaturated aqueous sodium bicarbonate then extracted 3×250 mL DCM. Thecombined organics were rinsed with brine, and dried over anhydroussodium sulfate before concentration in vacuo. The crude solid wasrecrystallized in multiple batches from boiling DCM/hexanes to afford8-[4-(dimethylamino)-2-ethoxy-phenyl]-7,8-dihydro-5H-[1,3]dioxolo[4,5-g]quinolin-6-one(2.27 g, 6.41 mmol, 90.47% yield). Notes: Off-white powdery solid,slightly hygroscopic.

¹H NMR (500 MHz, Chloroform-d) δ 7.79 (s, 1H), 6.73 (d, J=8.4 Hz, 1H),6.47 (s, 1H), 6.35 (s, 1H), 6.27 (d, J=2.4 Hz, 1H), 6.22 (dd, J=8.4, 2.4Hz, 1H), 5.88 (m, 2H), 4.49 (t, J=7.1 Hz, 1H), 4.11-3.99 (m, 2H),2.94-2.75 (overlap, 7H), 2.77 (dd, J=16.2, 6.4 Hz, 1H), 1.39 (t, J=7.0Hz, 3H). ¹³C NMR (126 MHz, Chloroform-d) δ 171.25, 157.27, 151.25,146.84, 143.62, 131.47, 128.77, 120.22, 117.83, 108.73, 104.88, 101.26,97.40, 97.38, 97.25, 77.42, 77.16, 76.91, 63.56, 40.86, 37.22, 35.35,15.09. IR (KBr): v (max) 3194, 3107, 2977, 2896, 1672, 1617, 1478, 1236,1041, 939, 798 cm⁻¹. HRMS: Exact mass calc'd for C₂₀H₂₂N₂O₄ requires m/z[M+H]⁺: 355.1658. Found: 355.1668. UV: (max) 219.888 nm. Melting point:N/A. Decomposes at 193° C.

Example 9: Biochemical Characterization of the Compound FQI-34

Analysis of Cell Viability—Cell Line GI₅₀.

QGY-7703 and A549 cells were cultured at 37° C. in 5% CO₂ in DMEM(Dulbecco's modification of Eagle's Medium; Corning) supplemented with10% Fetal Bovine Serum (FBS; Invitrogen). NIH-3T3 cells were cultured at37° C. in 5% CO₂ in DMEM supplemented with 10% Fetal Calf Serum (FCS;Atlanta Biologicals). For cell viability assays, 1,500 cells were seededin 96-well plates and treated with FQI-34 or vehicle (DMSO) atappropriate concentrations (DMSO at 1%) after 20 hours. After 72-hourincubation with compound or vehicle, cell growth was assessed via thePromega CellTiter 96® AQ_(ueous) One Solution Cell Proliferation Assay,a colorimetric method to determine the number of viable cells. 20 μL ofthe CellTiter 96® AQ_(ueous) One Solution Reagent was added directlyinto cultured wells and incubated for one hour, after which theabsorbance at 490 nm was read with a 96-well plate reader (Opys MRMicroplate Reader). The CellTiter 96® AQ_(ueous) One Solution containsan MTS tetrazolium compound which is bioreduced by cells into a coloredformazan product that is soluble in tissue culture medium. The quantityof formazan product as measured by absorbance is directly proportionalto the number of living cells in culture. GI₅₀ values were determinedfrom plots of the percentage of compound-treated cell growth to vehiclecell growth vs. compound concentration (GraphPad Prism; non-linearregression 4-parameter curve fit with variable slope). GI₅₀ values were0.26 μM in QGY-7703 cells, 1.8 μM in A549 cells and 1.49 μM in NIH-3T3cells (FIGS. 11A-11C)

Target Affinity Measured by Dual-Luciferase Reporter Assay; LSF IC₅₀:

NIH-3T3 cells were cultured at 37° C. in 5% CO₂ in DMEM (Dulbecco'smodification of Eagle's Medium; Corning) supplemented with 10% FCS (FCS;Atlanta Biologicals). Approximately 24 hours prior to transfection,200,000 cells were plated in 35 mm dishes. After 24 hours, cells were˜70% confluent, and were transfected for 5 hours with pEF1α-LSF, thereporter construct pGL3B-WT4E1b, phRLTK, and pEFGP (Grant, T. J. et al.Antiproliferative small-molecule inhibitors of transcription factor LSFreveal oncogene addiction to LSF in hepatocellular carcinoma. PNAS(2012), 109, 1-6). Vehicle (DMSO) or FQI-34 was then added, keeping theDMSO at 0.5%. Cell extracts were harvested 36 h post-transfection, andfirefly and renilla luciferase activities were measured via a dualluciferase assay (Promega Dual-Luciferase Reporter Assay System).Relative luciferase activity represents firefly luciferase activitynormalized to that of renilla luciferase in each extract. IC₅₀ valueswere determined from plots of normalized luciferase activity vs.compound concentration (Prism GraphPad; non-linear regression4-parameter curve fit with variable slope). LSF-Target affinity IC₅₀ was0.59 μM (FIG. 12).

Target Binding Assessed by Cellular Thermal Shift Assay (CETSA):

Drug-target engagement was assessed using CETSA as described by Molinaet al. (Monitoring Drug-Target Engagement in Cells and Tissues UsingCellular Thermal Shift Assay. Science (2013) 341, 84-87). QGY-7703 cellswere cultured at 37° C. in 5% CO₂ in DMEM (Dulbecco's modification ofEagle's Medium; Corning) supplemented with 10% Fetal Bovine Serum (FBS;Invitrogen). Cells were seeded into 10 cm plates and allowed to reach60-80% confluency. After trypsinization (0.05% Trypsin-EDTA,Invitrogen), cell pellet was washed twice with ice-cold 1×PBS (137 mMNaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.2). Cell pellet waslysed by addition of 200 μL freshly prepared ice-cold RIPA buffer (10 mMTris-Cl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodiumdeoxycholate, 0.1% SDS, 140 mM NaCl) supplemented with 1 mM Pefabloc(Sigma-Aldrich), and flash frozen in liquid nitrogen and thawed for atotal of three cycles. Cell lysate was centrifuged at 20,000×g for 20minutes to clarify. Supernatant was either treated immediately or savedat −80° C. for future use. 200 μL of the lysate was treated with FQI-34(addition of 2 μL 50 mM Stock; 500 μM final in 1% DMSO) or vehicle(DMSO) for 30 minutes at room temperature. After treatment, the lysatewas separated into 20 μL aliquots for thermal denaturation. Lysate washeated at the given temperature for 5 minutes, then placed on ice for 5minutes, and subsequently centrifuged at 20,000×g for 10 minutes toseparate soluble protein from aggregates. 10 μL of the supernatantcontaining the soluble protein was saved at −20° C. for immunoblotanalysis (anti-LSF and anti-LBP1a/b (Millipore); anti-GRHL1 (SierraSciences)).

Immunoblots were quantified using Image J and graphed using GraphPadPRISM. Area-under-the-curve (AUC) for the quantified western blots werecalculated with GraphPad PRISM and non-parametric t-test was calculatedto determine statistical significance between the curves. LSF is amember of a highly conserved family of transcription factors consistingof two branches: The LSF subfamily and the Grainyhead (GRH) subfamily.Despite a high degree of conservation in their DNA binding domains, andrecognizition of similar DNA motifs, the subfamilies target distinctsets of genes. CETSA results indicate FQI-34 directly binds the LSFsubfamily members LSF and LBP1a/b, however does not bind the relatedGRHL1 subfamily member (FIGS. 13A, 13B, 16A and 16B).

TABLE 5 QGY-7703 LSF ID GI50 (uM) LSF-reporter assay IC50 (uM) CETSAFQI-Br 0.65 1.823 YES FQI-F 1.50 NA NO FQI-Cl 0.31 1.732 YES FQI32 5.49Not tested NO FQI33 No inhibition Not tested NO >10 uM PEG-FQI Noinhibition Not tested NO >10 uM FQI34 0.26 0.7257 YES

Kinetic Solubility:

Kinetic solubility was measured via a turbidity based assay method(Kerns, E. et al. Automation in Pharmaceutical Profiling. Journal ofLaboratory Automation (2005) 10, 114-123). 198 μL of 1×PBS (137 mM NaCl,2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.2) was added to the wellsof a 96-well plate. FQI-34 (either dried or air-exposed (9% water asdetermined by NMR)) was dissolved in DMSO for 30 mM final concentrationimmediately before testing. The 30 mM stock was further diluted in DMSOappropriately for the desired final concentration to be tested in thesolubility assay. 2 μL of the DMSO solutions were added to the 96-wellplate such that DMSO was kept to 1%. The plate was sealed and gentlyshaken, and allowed to incubate at 37° C. for 2 hours, after which theabsorbance at 620 nm was taken. The concentration at which theabsorbance was greater than or equal to 0.01 absorbance units was takenas the precipitation point. Air-exposed FQI-34 first showed precipitatesat 100 uM while dry FQI-34 first showed precipitates at 150 uM (FIG. 14)

Example 10: In Vivo Bio-Analysis

Pharmacokinetic Analysis in Rat Plasma:

Studies were conducted at Cyprotex (Watertown Mass., www.cyprotex.com).The purpose of the study was to measure the concentration of FQI-34 inrat plasma samples. The study was performed under non-GLP conditions.All work was performed with appropriate local health regulation andethical approval. Plasma samples were taken at 0.08, 0.25, 0.5, 1, 2, 4,8, and 24 hours. Plasma samples were crashed with three volumes ofmethanol containing an analytical internal standard(propranolol/bucetin/diclofenac). Samples were then centrifuged toremove precipitated protein, and the supernatant was analyzed byLC-MS/MS using a Waters Xevo TQ mass spectrometer coupled with an AcuityHPLC and a CTC PAL chilled autosampler, all controlled by MassLynxsoftware (Waters). The analyte signal was optimized prior to analysis.After separation on a C18 reverse phase HPLC column (Waters Acquity HSST3 2.1×50 mm 1.8 μM) using an acetonitrile-water gradient system, peakswere analyzed by mass spectrometry (MS) using ESI ionization in MRMmode. All plasma samples were compared to a calibration curve preparedin rat blank plasma.

Male Sprague-Dawley rats were obtained from a commercial source andhoused in an AAALAC-approved facility until dosing. For IV dosing,FQI-34 was dissolved in 10% ethanol/16% PEG-400/74% saline at 0.4 mg/mLand in a 1% methylcellulose/0.5% Tween-80 suspension at 10 mg/mL for POdosing. Three rats per group were dosed by a single IV bolus (10 mL/kg)or PO (10 mL/kg). The rodent dosing scheme used is shown in Table 6.

TABLE 6 Dosing scheme for FQI-34 Pharmacokinetics of FQI-34-0l After IVor PO Dose in Rat Study#: NLS016-052516-01 Compound: FQI-34-0l DoseRoute: PO or IV Dose: 4 mg/kg IV; 100 mg/kg PO Date: May 25, 2016 DoseDose Rat B.W. voL. 5 15 30 1 2 4 8 24 Drug Group ID (g) (mL) time minmin min hour hour hour hour hour Delivery 1 A 242 2.4 8:11 8:16 8:268:41 9:11 10:11 12:11 4:11 8:11 4 mg/kg IV B 252 2.5 8:13 8:18 8:28 8:439:13 10:13 12:13 4:13 8:13 0.4 mg/mL C 223 2.2 8:15 8:20 8:30 8:45 9:1510:15 12:15 4:15 8:15 10 mL/kg 2 A 240 2.4 8:17 8:22 8:32 8:47 9:1710:17 12:17 4:17 8:17 100 mg/kg PO B 265 2.7 8:19 8:24 8:34 8:49 9:1910:19 12:19 4:19 8:19 10 mg/mL C 235 2.4 8:21 8:26 8:36 8:51 9:21 10:2112:21 4:21 8:21 10 mL/kg

Plasma PK-parameters were independently verified and interpreted usingPK-solver: An Excel add-In (Zhang, Y. et al. PKSolver: An add-in programfor pharmacokinetic and pharmacodynamic data analysis in MicrosoftExcel. Comput. Methods Programs Biomed. (2010) 99, 306-314), via anon-compartmental analysis using the linear-trapezoidal method (Fan, J.Pharmacokinetics. Biochemical Pharmacology (2014) 87, 93-120). Theplasma concentration-vs time curves are shown in FIGS. 15A and 15B andPK parameters are summarized in Table 7.

TABLE 7 Plasma PK Parameters. Rat Plasma PK Parameters IV: 4 mg/kg PO:100 mg/kg RAT A RAT B RAT C AVERAGE RAT A RAT B RAT C AVERAGE Bodyweight (g) 242 252 223 240 266 235 C₀ (ng/mL) 434 446 390 423 C_(max)(ng/mL) 545 548 564 552 500 501 577 526 T_(max) (hour) 0.5 0.25 0.5 0.424.0 2.0 2.0 2.7 AUC_((0-∞)) (ng/mL*hr) 1019 759 1028 935 4902 4026 59364955 T_(1/2) (hour) 0.73 0.72 0.99 0.81 CL (mL/min/kg) 65 88 65 73V_(Dss) (L/kg) 4.6 5.4 5.3 5.1 % Bioavailability 21% 17% 25% 21% F

Rat Tolerability:

Studies were conducted at Cyprotex (Watertown Mass., www.cyprotex.com).The purpose of the study was to determine the tolerability of FQI-34 inrats as administered by IV at 3 dose levels-5, 15, and 50 mg/kg. FQI-34was formulated in 20% DMSO/50% PEG-400/30% saline, and administered byIV at 5 mL/kg for compound or 10 mL/kg for vehicle alone. 6 rats perdose arm were used (3 male and 3 female), with single doseadministration with monitoring over 14 days. Gross necropsy wasperformed at the end of the study.

Observations:

Upon dosing, all rats receiving 50 mg/mL dose level experiencedpronation, increased respiratory rate, and decreased activity. However,within 30 minutes the rats returned to normal and experienced noabnormal behavior for the remainder of the study. Male Rat B died uponreceiving the 15 mg/mL dose, however this was believed to be due tostress and not compound related. All other rats at this dose levelremained normal throughout the study. Excluding the male rat B, all ratsstayed within normal limits for duration of study. Gross necropsy showedno abnormal findings.

TABLE 8 Summary of activity data QGY-7703 LSF-reporter Was CETSACompound GI50 (μM) assay IC50 (μM) performed? FQI-Br 0.65 1.823 YESFQI-F 1.50 2.905 YES FQI-Cl 0.31 1.732 YES FQI-32 5.49 Not tested Nottested FQI-33 >10 μM Not tested Not tested PEG-FQI >10 μM Not tested Nottested FQI-34 0.26 0.7257 YES

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SEQUENCE LISTING: SEQ. ID NO: 1    1gacgtcatgt tcgccgggca ggcgaggaaa gaggacgcca tgattggttg gcgctggggc   61ggcggacggt ggaagggcct ggcgagtcta ggttttacgc ctgtgctgga ctttctcctt  121ccatgtttcc aggccgtggg gggctacaga gggcgagaag tcggctcagc ggaaacctgg  181atttggttct aagccgtggg gttgagaagg ggtgaccgga agtgatcgtg ggactgaccg  241gaagcgaggc ctggagggga aagagagagc gagacctggg agggaggggg cctccagcag  301aaaggggcgg gggaaaaggt gcaaaagcag cgtgggagcg ccgggctggc ttcctgcggc  361tgctgctggt ctgactggga agcagcaagc caccactacg aactctcaag aggagtggga  421gtgcgggagt ccagagctgc ctctgggaag tctgcagtag ttgagcaaag gggtcctcac  481gttcctgaga gctgggcagg ggggattttg gaacctgggg cagccaagaa cgagcagcca  541agggtacggg agattagttg tgcacagagc agtgctggtc gggcttgggg gtggctggtg  601ggcactgcgt gggaaacctt ggtttgtagt tttcttggtt tgcgttactc ctgttgggta  661gaattaccct ccgcgccttt gtacaagaca cggtgtctcc tggggcaagg aaggagccag  721gatggcctgg gctctgaagc tgcctctggc cgacgaagtg attgaatccg ggttggtgca  781ggactttgat gctagcctgt ccgggatcgg ccaggaactg ggtgctggtg cctatagcat  841gagtgatgtc cttgcattgc ccatttttaa gcaagaagag tcgagtttgc ctcctgataa  901tgagaataaa atcctgcctt ttcaatatgt gctttgtgct gctacctctc cagcagtgaa  961actccatgat gaaaccctaa cgtatctcaa tcaaggacag tcttatgaaa ttcgaatgct 1021agacaatagg aaacttggag aacttccaga aattaatggc aaattggtga agagtatatt 1081ccgtgtggtg ttccatgaca gaaggcttca gtacactgag catcagcagc tagagggctg 1141gaggtggaac cgacctggag acagaattct tgacatagat atcccgatgt ctgtgggtat 1201aatcgatcct agggctaatc caactcaact aaatacagtg gagttcctgt gggaccctgc 1261aaagaggaca tctgtgttta ttcaggtgca ctgtattagc acagagttca ctatgaggaa 1321acatggtgga gaaaaggggg tgccattccg agtacaaata gataccttca aggagaatga 1381aaacggggaa tatactgagc acttacactc ggccagctgc cagatcaaag ttttcaagcc 1441caaaggtgca gacagaaagc aaaaaacgga tagggaaaaa atggagaaac gaacacctca 1501tgaaaaggag aaatatcagc cttcctatga gacaaccata ctcacagagt gttctccatg 1561gcccgagatc acgtatgtca ataactcccc atcacctggc ttcaacagtt cccatagcag 1621tttttctctt ggggaaggaa atggttcacc aaaccaccag ccagagccac cccctccagt 1681cacagataac ctcttaccaa caaccacacc tcaggaagct cagcagtggt tgcatcgaaa 1741tcgtttttct acattcacaa ggcttttcac aaacttctca ggggcagatt tattgaaatt 1801aactagagat gatgtgatcc aaatctgtgg ccctgcagat ggaatcagac tttttaatgc 1861attaaaaggc cggatggtgc gtccaaggtt aaccatttat gtttgtcagg aatcactgca 1921gttgagggag cagcaacaac agcagcagca acagcagcag aagcatgagg atggagactc 1981aaatggtact ttcttcgttt accatgctat ctatctagaa gaactaacag ctgttgaatt 2041gacagaaaaa attgctcagc ttttcagcat ttccccttgc cagatcagcc agatttacaa 2101gcaggggcca acaggaattc atgtgctcat cagtgatgag atgatacaga actttcagga 2161agaagcatgt tttattctgg acacaatgaa agcagaaacc aatgatagct atcatatcat 2221actgaagtag gagtgcggcg tttcgtgccc agtggctgct ccttccttca cctctgaaaa 2281cggccctctt gaagggggat atgaatggag atttgaaggt ctgcaagaac ctgactcgtc 2341tgactgtgtg tggaggagtc caggccatgg aggcagaatc ctggccctct gtgttggccc 2401aagctcttgt ggtacacaca gattactgcc caatatgcag ttctgcagct gttttagtta 2461aatttctgga ccttgttgtt gttaaatatc agtagaaact ctacataatt tagagtgtat 2521gtagggcata atgatgatgg gaattgtgtg atgtttaaca ggaagatctt aaattttgtg 2581atatggagcc ctgtaatttt tttcttatat aaaaatgggt atctatattc ataagactag 2641gtcttcaatc tctttgtact ggtctcaaat gtactggtat ctctgctttt tgccacagtt 2701tgtccctgaa aactttatca gtgaggagaa atacagaatt ttccttttgg ttcccctgta 2761ataccacaac taatagtatt ttcagctaac atttattgac caggcagtgt gataaatttt 2821ttgaatgacc atcttgttta accccctaat cacactatct gaagtaggtg ctatagtgac 2881ccccaaccga agatgaggaa atggagacac acagtggcta agtagcttgc taggtgccag 2941gagctggcag gcagtgatgc tgaaatccaa accaggcaat ctggccctcc ttattcaccc 3001tctcatacca ccagtgctac ccattataac ctgtgttcac ttcgcttatt actgtggccc 3061ttctggtgac tacaacataa cactgagtat gaactgagga attaaccact gaaagagaca 3121aagcacacct aaataatgat gaacaaatca agacctacaa gaaatgaaca catagatgct 3181ttttaagtgt cattagttat cggaatcaaa gaatttgagg tggtgacaac tggggattat 3241ctgtaactcc ccaaatttac tgttcttgaa tgacattctc aaactattta ttaatgagtt 3301agtctcatcc ctgctttctg tttcctctct ctgcatcttc tcttggcata ggaagttatt 3361ttactcatgc tatgtttttt aatagaagct ttacaaaaaa agatgcattc ccttcctgtt 3421cacactctta ccatgttttt gttcttcctt aagaatgatt gtgtgtgtgt gtgtgtgtgt 3481gtgtaagtgt gtaagtgtaa catcatgggt tttctttctt cacttgtaaa gttttatcta 3541gaaatacttt tacaggttat gttgaggtac acaaagataa tgttctaaga ttgtgtattg 3601gatttggatt tgtgtgtgtg tgtgtgtgtg tgtgtgttta aagcatttga taaggtttaa 3661atgtttttta tacagagatt tttgttcatt aaactcaatc tgcatcatgg tctaaSEQ ID NO: 2    1mawalklpla deviesglvq dfdaslsgig qelgagaysm sdvlalpifk qeesslppdn   61enkilpfqyv lcaatspavk lhdetltyln qgqsyeirml dnrklgelpe ingklvksif  121rvvfhdrrlq ytehqqlegw rwnrpgdril didipmsvgi idpranptql ntveflwdpa  181krtsvfiqvh cisteftmrk hggekgvpfr vqidtfkene ngeytehlhs ascqikvfkp  241kgadrkqktd rekmekrtph ekekygpsye ttiltecspw peityvnnsp spgfnsshss  301fslgegngsp nhqpeppppv tdnllptttp geaqqw1hrn rfstftrlft nfsgadllkl  361trddviqicg padgirlfna lkgrmvrprl tiyvcqeslq lreqqqqqqq qqqkhedgds  421ngtffvyhai yleeltavel tekiaqlfsi spcgisqiyk qgptgihvli sdemignfge  481eacfildtmk aetndsyhii lk SEQ ID NO: 3 5′-ACACGCTTATGCGGGTATGT-3′SEQ ID NO: 4 5′-GAACATTTGGTAGGGGGAAA-3′ SEQ ID NO: 5TGGCTGGTTATGGCTGGTCAGACTAG

What is claimed is:
 1. A method for reducing cell proliferation in acancer that overexpresses LSF in a subject, the method comprisingadministering an effective amount of a compound of Formula (III′) to asubject in need thereof, wherein the Formula (III′) has the structure:

wherein: R^(1′) is an aryl substituted with at least one C₁-C₆ alkoxyland at least one di(C₁-C₂₄alkyl)amino, halogen or C₂-C₈alkenyl; R² andR³ are hydrogen; R⁴ is hydrogen; R⁵ is hydrogen; R⁶ and R⁷ are hydrogen;R¹⁰ and R¹¹ are hydrogen; and pharmaceutically acceptable salts thereof,and wherein the compound is capable of inhibiting late SV40 factor(LSF).
 2. The method of claim 1, wherein the cancer is hepatocellularcarcinoma (HCC).
 3. The method of claim 1, wherein the cancer is a solidtumor and is selected from the group consisting of breast cancer, colorcancer, ovarian cancer, lung cancer, kidney cancer, cancers of thehematopoietic system, cancers of the endometrium, cervical cancer,cancers of the upper digestive tract, stomach cancer, liver cancers andcancers of the small intestine.
 4. The method of claim 1, furthercomprising selecting the subject with hepatocellular carcinoma prior toadministering the compound or the subject has been identified to have ahigher expression of LSF in the liver, as compared to a reference level.5. The method of claim 1, wherein the compound is selected from thegroups consisting of


6. The method of claim 1, wherein the compound is FQI-34.
 7. The methodof claim 1, wherein the cancer is liver cancer and in Formula (III′):R^(1′) is an aryl substituted with at least one C₁-C₆ alkoxyl and atleast one di(C₁-C₂₄alkyl)amino or halogen; R², R³, R⁴, R⁵, R⁶, R⁷, R¹⁰and R¹¹ are hydrogen.
 8. The method of claim 7, wherein R^(1′) is anaryl substituted with at least one C₁-C₆ alkoxyl and at least onedi(C₁-C₂₄alkyl)amino.
 9. The method of claim 1, wherein the cancer isliver cancer and the compound is FQI-34.
 10. The method of claim 1,wherein the cancer is liver cancer.
 11. The method of claim 1, where R¹is a phenyl substituted with one C₁-C₆ alkoxyl and one ofdi(C₁-C₆alkyl)amino, halogen or C₂-C₆alkenyl.
 12. The method of claim 1,wherein R¹ is

where R²¹ is C₁-C₆alkyl; R²² and R²³ are independently C₁-C₆alkyl; R²⁴is Br, F, or Cl; and R²⁵ is C₂-C₆alkenyl.
 13. The method of claim 12,wherein R¹ is


14. The method of claim 13, wherein R¹ is